A review of small animal dosimetry techniques: image-guided and spatially fractionated therapy

Research in small animal radiotherapy is a crucial step in clinical translation of novel radiotherapy techniques, either delivered as stand-alone treatment or in combination with other treatments, such as chemotherapy and immunotherapy. In order to efficiently translate preclinical findings to the clinical setting, preclinical radiotherapy must replicate clinical therapy in terms of mode of delivery as well as dose delivery accuracy as closely as possible. In this review article, we focused on the description of dosimetry tools for radiotherapy of small animals delivered with kilovoltage x-ray beams on image-guided irradiators and in a spatially-fractionated manner by means of microbeam therapy. The specifics of dosimetry of kilovoltage x-ray beam deliveries with small, often sub-millimeter, beams are highlighted, and suitable dosimeters, phantoms, and dose measurement and calculation techniques are reviewed. Future directions for accurate real-time high spatial resolution dosimetry of small animal irradiations are also discussed.


Introduction
Cancer radiotherapy research performed with small animals in a preclinical setting is crucial in advancing patient cancer care.It is used to not only evaluate novel radiotherapy techniques, alone or in combination with chemotherapy or immunotherapy, but also to evaluate imaging techniques to assess radiotherapy treatment outcomes.
Compared to clinical irradiations, small animals are typically irradiated with much smaller fields of 1-10 mm in size.Small field dosimetry has been in the frontline of dosimetry research for a about a decade.While preclinical radiotherapy research has been investigated with proton beams and other particles, preclinical radiotherapy is most often delivered with kilovoltage (kV) x-ray beams, i.e., with beams of lower energies compared to clinical radiotherapy performed with megavoltage photon beams [1].Such difference in beam energy also poses challenges to small animal irradiation dosimetry.The focus of the review article will be on dosimetry of radiotherapy of small animals performed with kilovoltage x-ray beams, delivered either with image-guided or single-field irradiators, or in a spatially-fractionated manner.

Small animal radiotherapy
Since the 1970s cabinet irradiators have been used as a small scale, cost-effective tool in preclinical research [2,3].Current commercial irradiators for the radiation therapy of small animals includes the PXi X-RAD 320 (Precision X-Ray Inc., North Brandford, CT), Xstrahl RS225 and RS320 (Xstrahl Inc, Suwanee, GA), Faxitron MultiRad 160, 225, and 350 (PharmaMedSci, St. Laurent, Quebec), Gammacell 40 (Best Theratronics, Ottawa, Ontario), Pantak Therapax-150 and DXT-300 (Pantak Inc., Branford, CT).These irradiators usually treat with only a single stationary field with very simple collimation.In addition, they inherently lack the ability for precision radiation therapy as they do not contain on-board imaging technology to accurately plan and deliver conformal treatments, and are mainly used for general flank tumor, total body irradiation (TBI), and cell irradiations [4,5].Irradiation times are usually approximated from simple in-phantom measurements where custom made jigs and lead blocks must be created to provide more conformal and specialized treatments [6][7][8][9].A higher degree of treatment accuracy can be achieved with medical linear accelerators (LINACs), currently commercially produced by Varian and Elekta as the two largest LINAC manufacturers.While LINACs are excellent at performing conformal irradiations, they can cost upwards of a million dollars and are expensive to maintain, require specialized training to operate, and are specifically calibrated to treat humans.
A shift in preclinical research away from standard cabinet irradiators began in the mid 2000's when the first image-guided small animal irradiator was created, creating for the first time, conformal preclinical radiation therapy on a practical and small scale [10][11][12][13][14][15].This shift allows for more precise and accurate treatment delivery that includes treatment planning systems (TPS) and onboard image guidance as a small scale analogue to a LINAC.This introduces the ability to accurately treat tumours implanted within the animal, and increases the geometric dose delivery accuracy to within 0.2 mm. 11,16

Spatially-fractionated radiotherapy
Spatially-fractionated radiotherapy (SFRT) delivers very small beams of high doses interleaved with areas of low doses (Figure 1).It was first demonstrated to protect normal tissues in brain following irradiations with 22 MeV deuterons [17].Curtis et al. observed no tissue necrosis developed in the normal mouse brain after focal administration of >1 kGy along the peak dose areas of microbeam arrays [17].SFRT can be delivered with kV x-rays as microbeam radiation therapy (MRT) with beams of ~25-100 µm in size with centre-to-centre (c-t-c) distances ~200-400 µm gaps [18,19] or as minibeam radiation therapy (MBRT) with 500-700 µm beams and ~mm-long c-t-c distances [20].Xray MRT delivered with synchrotron beams has shown to reduce normal tissue toxicity in brain tumors in rats [21] and mammary carcinoma in mice [22].MRT and MBRT are characterized by a number of dosimetry parameters, such as the peak doses, the valley doses, and the peak-to-valley ratios (PVDRs).A recent review paper summarized the history, current status, and future prospects of SFRT [23].To this date, however, it is not clear what are the dosimetric parameters driving the reduced normal tissue toxicity in SFRT and it is therefore important to evaluate these parameters in radiobiological studies accurately.

Image guided irradiators
Two commercial image-guided small animal irradiators are currently available, the PXi X-RAD SmART and the Xstrahl Small Animal Radiation Research Platform (SARRP).Both units are entirely self-shielded, contain a TPS, and include on-board image guidance.The PXi X-Rad SmART operates at a 220 kVp tube voltage, 13 mA tube current, 0.8 mm Be internal and 0.30 mm Cu external filtration, 20° tungsten anode, and a 3.0 mm focal spot for therapy beam delivery.The maximum uncollimated dose rate at a 30 cm isocenter distance in 2 cm of water is approximately 4 Gy/min.Treatment collimators range from 1 to 25 mm circular and 5 × 5 to 40 × 40 mm 2 square field sizes at isocenter.CBCT images are obtained using a stationary couch and 360° rotating source and flat-panel detector, operating at a common imaging protocol of 60 kVp tube voltage (40 to 100 kVp range), 0.200 mm pixel size, 1 mA tube current, 5 frames per second (fps), and 60 s scan time, using a 2.0 mm Al external filtration with a 0.4 mm focal spot [16].
The Xstrahl SARRP operates at a 220 kVp tube voltage with a 13 mA tube current, 0.8 mm Be internal and 0.15 mm Cu external filtration, 20° tungsten anode, and a 3.0 mm focal spot for therapy beam delivery.The maximum uncollimated dose rate at a 35 cm isocenter distance in 2 cm of water is approximately 3.6 Gy/min.Treatment collimators range from circular 0.5 to 40 mm and 3 × 3 to 10 × 0 mm 2 squared field sizes at isocenter.CBCT images are obtained using a stationary source and flatpanel detector with a 360° rotating couch, operating at a common imaging protocol of 60 kVp tube voltage (40 to 80 kVp range), 0.200 to 0.350 mm pixel sizes, 0.8 mA tube current, 6 fps, and 60 s scan time, using a 1.0 mm Al external filtration with a 0.4 mm focal spot [10][11][12].Imaging dose using the common imaging protocol is approximately 3.5 cGy to the entire body of a 20 g mouse [24,25].

Synchrotron beams
A synchrotron is a type of cyclical particle accelerator where a beam of charged particles are accelerated through a changing electromagnetic field in circular or oval paths that can be used to generate x-ray beams with dose-rates up to 5,000 times higher than clinical radiotherapy [26].Synchrotrons deliver highly-collimated quazicontinuous polychromatic x-rays with mean energies of 30-500 keV, therefore of higher energies compared to image-guided small animal irradiators.For example, the synchrotron beam at the European Synchrotron Research Facility (ESRF) in Grenoble is 50-µm thin, has a mean energy of 102 keV, and delivers extremely high dose rates of 16 kGy/s [27].An overview of available synchrotron beamlines and their properties can be found in a recent publication by Montay-Gruel et al [28].The high dose-rate available in synchrotron beams can be used in combination with MRT allowing for spatially fractionated dose using an array of parallel beams of 25-100 µm widths to deliver tumoricidal dose while providing normal-tissue sparing effects [29][30][31].
Synchrotrons lend themselves to small animal applications for therapeutic radiation therapy such as microbeam dosimetry, FLASH, establishing tumour and normal tissue affects in small animals, generating genetic models such as epilepsy and human tumour models in small animals, and the study of brain function with microbeam irradiation [30][31][32][33][34][35], including synchrotrons used for small animal imaging [36,37].These synchrotron beams can be generated by either large-scale beam-lines [30,34,35,38], or more recently, through compact laboratory-sized synchrotrons for lower kilovoltage x-ray beam energies [32,36].

Ionization chambers
Ionization chambers are the gold standard in reference dosimetry for kV beams for delivering a high level of accuracy, reproducibility, sensitivity, and real-time output.This is especially true for cylindrical ionization chambers as these chambers have a nearly constant energy response between the 40 and 300 kV tube voltages used in image-guided small animal irradiators [39].In North America, the American Association of Physicists in Medicine Task Group 61 (AAPM TG-61) is one of the most used dosimetry protocols and recommends that absolute dosimetry and beam quality be exclusively obtained with air-filled ionization chambers that are calibrated by an Accredited Dosimetry Calibration Laboratory [39].For relative dosimetry, parallel plate ionization chambers and smaller volume cylindrical ionization chambers may also be used.
Since image-guided small animal irradiators require small fields (≤ 4 cm), small volume cylindrical ionization chambers are utilized to prevent volume averaging effects.PinPoint ionization chambers can be 0.015 cm 3 in volume (2 mm diameter, 5 mm length) with a 0.20 nC/cGy response and are well suited for field sizes as small as 2 × 2 cm 2 (as specified by the manufacturer, PTW) to avoid volume averaging effects.Multiple studies show an uncertainty greater than 6% when measuring 5 mm diameter field sizes, and an uncertainty within 3% when measuring field sizes 6 mm and larger when using PinPoint ionization chambers [40,41].In a study by Charles et al [42], it was determined that a 0.015 cm 3 volume PinPoint ionization chamber had an uncertainty in output factor measurements for small field sizes of approximately 1.0 and 3.5% for 1.5 and 1.0 cm diameter field sizes, respectively, however this analysis was completed with a 6 MV beam.Hill et.Al. recommends using a PinPoint chamber with an aluminum electrode, as a PinPoint chamber with a steel electrode reported over responding by as high as 8% due to the photoelectric effect from the high atomic number of steel [43].Compared to the larger volume ionization chambers for absolute dosimetry, a 0.6 cm 3 volume (6 mm diameter, 23 mm length) with a 20 nC/cGy sensitivity can be used for field sizes down to 4 × 4 cm 2 .If smaller field sizes are to be measured, diamond detectors, small scintillators, or radiochromic film should be used.

Radiochromic films
One of the greatest challenges in small animal dosimetry is the high spatial resolution required to measure small fields, especially when employing spatially fractionated microbeams, and this is where radiochromic films are second to none.The most widely used and characterized radiochromic film for kilovoltage dosimetry currently is GAFChromic EBT3 film (Ashland Advanced Materials, Bridgewater, NJ) [44][45][46][47][48][49][50][51][52][53][54][55].EBT3 film has a nominal 28 μm thick active layer (Li-PCDA with dominant peaks at 633 and 595) [56] sandwiched between 125 μm thick matte layers of polyester-substrate, with an optimal dose range between 0.20 and 10 Gy and ≤ 25 μm measurement resolution [57,58].This makes EBT3 film optimal for measuring small field output and profiles down to a 25 μm field size.For doses up to 40 Gy, EBT-XD should be used [59].For FLASH and/or microbeam dosimetry, GAFchromic HD-V2 (Li-PDCA active layer with dominant absorption peaks of 670 and 635 nm) can be used as this film has a dose range between 50 to 1000 Gy and an extremely high spatial resolution of ≤ 5 μm [56,60,61].Vaiano et al [53] developed a method to use EBT3 film up to 100 Gy with a 4% reading uncertainty for 250 kVp x-rays.
For an increase in dosimetric accuracy, it is recommended that a triple-channel calibration (red, green, and blue channels) be used over single-channel (red channel) and double channel calibration methods, including creating a separate calibration for each film batch and given beam quality [56,62].A number of triple-channel calibration techniques are available [62][63][64].Using a triple channel method can extend the dose range of EBT3 film to 40 Gy [44].The red channel is most sensitive for the lower energy range and the green channel is most sensitive for the higher energy range of radiochromic film, and the blue channel provides a response signal to automatically correct for nonuniformities in the radiochromic film [44].For the reading of radiochromic film, it is recommended to use a professional colour flatbed scanner (such as the Epson 10000XL), circular ROIs, 600 dpi for small field dosimetry, RGB colour mode, ≥ 16 bit depth, and saved in an uncompressed .TIFF format [56].Great care must be exercised in the calibration, measurement, handling, and reading of radiochromic film to produce accurate dosimetry, especially kilovoltage dosimetry (due to energy sensitivities), and readers are directed to the recently updated AAPM TG-235 report on radiochromic film dosimetry [56].However, for SFRT, flatbed scanners might not be sufficient to resolve the peak and valley doses due to microscopic spatial inhomogeneities that arise due to the granular structure, defects, and compositional variations in the film.This can be mitigated by using a microscope for improved resolution scans in microbeam dosimetry [61].

Plastic scintillator detectors
Plastic scintillators have been used in megavoltage therapy, with a growing interest in examining their properties for kilovoltage energies [65][66][67].Recently, plastic scintillators have been characterized and successfully validated for use in the 40 to 220 kVp tube voltage energy range within multiple small animal irradiators [49,[68][69][70][71][72].Due to an energy dependence that is present for energies below 250 kVp, output must be corrected for by using a combination of mass-energy absorption coefficients, Monte Carlo (MC) obtained beam spectra information, and quenching corrections [66,71].
The greatest advantage of plastic scintillators lies in their high special resolution to measure very small fields and profiles down to a 3 x 3 mm 2 field size [49].This is possible due to a very small 1 mm 3 active volume of polystyrene.While radiochromic film can measure even smaller field sizes in 2D, the advantage of plastic scintillators over film is that they can measure output in real-time.This includes the ability for real-time in-vivo mouse measurements with an average accuracy within 4.2% when compared to TPS calculations as shown by Le Deroff et al [68].Esplen et al [72] acquired point dose measurements inside a heterogeneous mouse phantom with field sizes between 3 x 3 and 10 x 10 mm 2 and reported accuracy within 3.2% when compared to MC simulations.Johnstone et al [49] acquired in-air profiles using 3 × 3 and 5 × 5 mm 2 field sizes and reported a mean 2.4 % difference inbetween the penumbra region when compared to radiochromic film measurement and MC simulations, but found that measuring a 1 mm diameter profile with the scintillator under responded by 20% due to volume averaging effects.More recently, plastic scintillators have been demonstrated to be a suitable dosimeter for 120-kVp ultrahigh dose-rate experiments performed with a modified x-ray tube [73,74].

Diamond detectors
A real-time commercially available detector with the ability to measure field sizes smaller than PinPoint ionization chambers are diamond detectors.The properties of an ideal detector system are high reproducibility, sensitivity, linearity, spatial resolution, and tissue equivalence as well as low dose rate, angular, and energy dependence, and diamond detectors are excellent for these categories [75].Natural and synthetic diamond detectors have approximate tissue equivalence, with a push towards synthetic chemical vapor deposition (CVD) due to the relatively availability, low cost and dose-rate linearity compared to natural diamond detectors [76].
A common commercial synthetic diamond detector is the PTW 60019 microDiamond (PTW, Freiburg, Germany) that has a small nominal 0.004 mm 2 sensitive volume, 1.1 mm radius, 0.001 mm thickness, waterproof, contains a nominal 1 nC/Gy response, and conveniently has a 0 V bias [75].This detector has been characterized for kilovoltage energies between 40 and 250 kVp, compared to other commonly used detectors, and achieves good dose-rate linearity, but requires pre-irradiation and has an energy dependence below 250 kVp, especially below 60 kVp [77].
Diamond detectors are excellent for small field dosimetry of kilovoltage x-ray beams when energy dependence is accounted for, including measuring profiles in spatially fractionated x-ray beams of 30-120 kVp, up to 700 Gy/s dose rates, using 600 µm minibeams using the PTW 60019 microDiamond detector [26].Other studies have also used microDiamond detectors to measure small field size output and profiles from 2 cm down to 2 mm for < 300 kVp x-ray beams [78,79].Small fields have also been measured with diamond detectors in image-guided small animal irradiators [80,81].

Other detectors
Other small-volume detectors are also suitable for dosimetry of small animal radiotherapy.Their sizes must be smaller than the irradiation field and their response to kV x-ray beams must be carefully characterized.Thermoluminescent detectors (TLDs) are popular detectors for small animal radiotherapy due to their small size down to 1×1×1 mm 3 microcubes [82,83].They can be conveniently used for in vivo dosimetry [84].TLDs can also be inserted into cadavers for accurate dose measurements mimicking experimental conditions for mouse brain [85] and bone marrow irradiations [86].TLDs were used for a mail audit to verify the accuracy of small animal dosimetry by the University of Wisconsin Medical Radiation Research Center [87] and MD Anderson Cancer Center [88].TLDs overrespond to low-energy x-rays (< 3 mm Cu HVL) by as much as 13% and need to be carefully characterized in order to achieve dosemetric accuracy [89].Injectable 0.6-mm long TLD rods have also been developed for small animal radiotherapy.They were injected subcutaneously in the dorsal neck of mice irradiated in a Cs-137 irradiator [90].
Optically stimulated luminescent detectors (OSLDs) would be suitable due to their small size, unfortunately their high energy dependence limits their use for pre-clinical applications.For example, nanoDot OSLDs are 0.2-mm thick disk-shaped detectors with a diameter of approximately 5 mm encased in a light-tight 10 × 10 × 2 mm 3 plastic carrier overrespond by 85% in a 2-mm Al HVL beam compared to a 9.5-mm Al HVL beam [91].The nanoDot OSLDs have been shown by Poirier et. al. to have a strong variation in light output in the 40-300 keV range that over responds by as much as a factor of 4 compared to a 6 MV photon beam, including a 15% variation in dose between different xray units of equivalent effective energies [92].For accurate dosimetry, OSLDs must be well characterized for a given beam quality and irradiation geometry in order to account for their energy dependence.Similarly, microMOSFET dosimeters are of a small physical size of 1×1 mm 2 and 3.5 mm long but exhibit large energy dependence.Their overresponse to a 2 mm Al HVL beam compared to a 6 MV photon beam is 400% [93].

Homogeneous phantoms
Solid water is the phantom type of choice for kilovoltage x-ray reference dosimetry in small animal irradiators for its practicality and water equivalence.The phantom must be water equivalent or the kilovoltage absorbed dose measurements acquired will not adequately represent dose to water and can resulting in incorrect dose calculations of as much as 6% [94].The ICRU Report 44 recommends for solid water to be considered water equivalent; it must not introduce uncertainties to the absorbed dose greater than 1%, where a given thickness of phantom material should have the same radiation and scattering properties as the same thickness of water [95].This is important as there are a wide range of phantom materials being used without the exact material types specified within image-guided small animal irradiators [7, 10-12, 14, 15, 20, 24, 69, 96-111].Hill et al [112] examined depth doses and profiles for 10 commonly used solid water materials compared to water under low energy kilovoltage dosimetry (40 to 300 kVp) and recommends the most accurate materials (dose percentage differences less than 2% to water) to be RMI-457 (Gammex Inc., Wisconsin, USA), PWDT (CIRS, Virginia, USA), and Virtual Water (CMNC, Tennessee, USA).The worst performing solid water were plastics, namely PMMA, Polystyrene, and Plastic Water (CIRS), with percentage differences between 3-6% compared to water.To bypass this water equivalence issue, Arango et.al [40].used a small 334 × 336 × 422.5 mm 3 (L × W × H) 3D scanning water tank MP3-XS (PTW, Freiburg, Germany), which fit within the small confines of the self-shielded Xstrahl SARRP.Most users of small animal irradiators don't have access to a small scanning water tank, thus solid water phantoms are almost exclusively used.The venders of the image-guided small animal irradiators provide small 6 x 6 x 0.5 cm 3 plastic solid water slabs that fit into a custom jig that is attached to the head of the irradiator to be used with radiochromic film [24,109,113].Large solid water is provided for LINACs (i.e., large 20 × 20 cm 2 slabs) must be specially machined smaller to fit on the small animal treatment couch, or else the user will have to create a jig to raise up and support the larger solid water slabs from the bottom of the irradiator [25].Solid water slabs can be used with radiochromic film or machined for the insertion of ionization chambers for dosimetry measurements.Simple cylindrical homogenous mouse phantoms (i.e.~6 cm long and 2.5 cm diameter) that dosimeters such as PinPoint ionization chambers, OSLDs, and TLDs have been used for pre-irradiation dose verification [87,114,115].

Heterogeneous phantoms
While the material of solid water must be carefully chosen to match water equivalence, the dosimetric properties of heterogeneous materials for low kilovoltage photon beams such as bone in small animals are not well established, with material densities and elemental compositions shown to produce a significant absorbed dose difference compared to using ICRU44 human tissues [95,116,117].To accurately match the physical densities and elemental composition of mouse tissues to create mouseequivalent materials, it can be accomplished through elemental composition analysis methods such as x-ray fluorescence (XRF), atomic absorption spectroscopy (AAS), and inductively coupled plasma mass spectrometry (ICP-MS) [118][119][120][121][122].However, most small animal research using heterogeneous phantoms do not create custom mouse-equivalent materials and instead use human-equivalent materials (or non-tissue equivalent materials such as cork [8] and PMMA [87]) that may not be validated at low kilovoltage energies.The most accurate way to validate the dosimetric properties of heterogeneous materials is to compare experimental measurement with TPS or Monte Carlo Simulations [24,117,[123][124][125][126][127][128][129].Validation measurements are usually carried out by placing radiochromic film adjacent to solid water slabs aside heterogeneous slabs such as bone and lung [24,123,124,130].Commercial heterogeneous materials can be obtained from companies such as Gammex and CIRS.Recently, a number of studies have replicated the physical geometry and material composition of mice by creating custom realistic heterogeneous and anthropomorphic mouse phantoms (Figure 2) [72,[131][132][133][134][135][136][137][138].These phantoms are useful in better replicating the actual radiation scattering events, geometry, and tissue attenuation of actual mice for treatment dose verification.These phantoms have been created using realistic mouse geometry that includes tissue materials such as bone, lung, brain, liver, heart, and kidney.Inserts for dosimeters within these phantoms include radiochromic film, TLDs, scintillators, and PinPoint ionization chambers.

Dose measurements
Many protocols exist for kilovoltage reference dosimetry such as the AAPM TG-61 [39] , however, as of this writing, no official protocol for reference dosimetry exists for standard cabinet and image-guided small animal irradiators.The limited geometry within the self-shielded irradiators do not allow for the scatter-free geometry recommended by the aforementioned protocols.Thus, a dosimetry protocol specifically for cabinet irradiators is needed.
In North America, the most common approach is following a modified version of the AAPM TG-61 protocol [14, 24, 25, 41, 49, 98-100, 103, 107, 110, 146-148].For absorbed dose to water measurements, the AAPM recommends the "in-air method" or the "in-phantom method" using an ADCL accredited cylindrical ionization chamber.The setup geometry and correction factors for each method are described in detail in the AAPM TG-61 protocol [39].As a result of setup and reproducibility difficulties using water phantoms within image-guided small animal irradiators, many studies used the in-air method [6,14,25,41,49,96,99,103,107].In this method, a large volume ionization chamber (i.e., 0.6 cm 3 volume) can be secured to the treatment couch and leveled at isocenter with the active volume extended off the edge of the couch to avoid couch backscatter, while using a collimated field no less than 4 cm in diameter for large ionization chambers.To establish the most accurate positioning possible, centering the active volume of the ionization chamber with the CBCT and not with the lasers has been done [99].An EPID detector can also be used to position the chamber in the x-and y-directions using a collimator for centering guidance, and a custom-built frontpointer can be used for positioning in the z-direction [25,149].The output may be acquired at the four cardinal angles to verify that the chamber is correctly positioned at isocenter, and to verify output with changing gantry angle.In the event that a small water phantom is available (i.e., 6 × 6 × 10 cm 3 to 30 × 30 × 30 cm 2 ) and can be positioned in a reproducible way, the in-phantom method can be used with a cylindrical ionization chamber placed at isocenter at a depth of 2 cm in water [40].
For daily pre-treatment output verification and monthly output measurements, it is recommended to measure air-kerma at isocenter to verify any changes in machine output over time, as this method allows for a quick, simple, and reproducible setup compared to in-phantom measurements with less risk for setup error [25].Ease of setup is of high importance as low kilovoltage x-rays have a high dose gradient with distance from the source, and a setup reproducibility on the order of 1 mm is required for accurate and reproducible dosimetry [25,139].For commissioning and annual dosimetry, the in-phantom method for output measurements should be used, with great care taken into the accuracy of the setup [96,103,110].Recently, the use of a built-in EPID imager has been examined as a means of simplified dosimetry within an image-guided small animal irradiator [149-151].
It is recommended to not use the reference dosimetry dose rate as a means to establish the irradiation time for therapy treatments for mice or cell irradiations if the irradiation geometry and mouse/cell-thickness is not identical to the reference dosimetry.The different scattering conditions can misrepresent the dose in mice by as much as 15% due to large differences in the lateral scatter, backscatter, and field size differences within the full-scattering conditions recommended by the AAPM TG-61 compared to the relatively small thicknesses of mice or irradiated cells [6,152,153].Instead, it is recommended to use a cylindrical solid water mouse phantom that houses a PinPoint ionization chamber to better model the realistic scattering conditions of mice treatments [114,115].If a mouse phantom is not available, the backscatter factors from Chen et al [153] that report scatter factors with varying phantom thickness can be used with reference dosimetry measurements, and likewise, could be used for quantifying the mm-thick backscatter conditions for cell irradiations.Beam quality for low kilovoltage x-rays is quantified by using both the first half-value layer (HVL) and kVp and are required properties for reference dosimetry [39,124,154].According to the AAPM TG-61, HVL should be measured under a scatter free and narrow-beam geometry by placing the ionization chamber approximately 100 cm from the source with collimation and attenuating material (Cu or Al) approximately 50 cm from the chamber.The collimation of the beam at 50 cm for narrow-beam geometry should create a field size just large enough to provide a uniform exposure across the sensitive volume of the chamber [39].This 100 cm geometry cannot be achieved within the small self-shielded confines of image-guided small animal irradiator cabinets, so modifications must be made.These modifications include placing a large cylindrical ionization chamber approximately 50 cm from the source, with the attenuating material and beam collimation placed as far from the detector as possibly [24,99,103,106].The distance of the chamber may be varied to allow at least 20 cm between the walls of the irradiator and the chamber to reduced beam scatter to the measurements [24,103].A three-point semi logarithmic interpolation technique has been recommended to determine HVL for kilovoltage energies as larger amounts of measurement points may inherently change the beam quality during HVL analysis [124].
Percentage depth dose (PDD), off-axis profile measurements, and relative output factors can also be obtained.These measurements are used for commissioning of the irradiator, quality assurance testing, and dosimetry for treatment planning calculations.Measurements can be performed with radiochromic film wedged in-between small solid water slabs placed on the treatment couch [110], or placed within a jig attached to the head of the irradiator [24,109,113].When placing solid water on the couch, care must be taken to prevent any bowing of the couch that will skew output readings [25].If a small scanning water tank can be setup in a reproducible way, a waterproof high resolution PinPoint ionization chamber can be used for PDD, profile, and output factor measurements [40,97,103].

Dosimetry accuracy and reproducibility
The accuracy and reproducibility of kV beam dosimetry is heavily dependent on quantifying the irradiation geometry.This includes the source-to-surface distance (SSD), the attenuation and material above the point of measurement, the backscatter thickness and material below the point of measurement, field size, and beam quality (kVp and HVL).In conformal small animal irradiators, the dose can change by 10% with the following: 2 cm change in SSD, 10% per cm of attenuating material [48,155], 0.05 mm change in Cu external filtration [154], or 1 cm of backscatter (field sizes > 1 cm) [153,156].Changes in dose from backscatter thickness become negligible at backscatter thicknesses of approximately 5 cm and thicker [153].A 5% change in dose can occur with a change in field size from 5 × 5 mm 2 to 3 × 3 mm 2 and to 10 × 10 mm 2 [24].An increase in field size from 1 to 4 cm increases the backscatter factor for mice irradiations by 10% [153].Changes in field size or beam quality affect the attenuation, backscatter, and overall dose [39,48,124,153,155,156], and changes in beam quality can affect the relative biological effectiveness (RBE) [154].The quantification of dose changes with irradiation geometry in a small animal irradiator are illustrated in Figure 3.An absorbed dose variation of only ±5% can affect tumour tissue control probability by 10-20% [1,157,158].This is of course valid for both clinical and preclinical radiotherapy and accurate dosimetry is of highly importance during the commissioning of clinical linear accelerators as well as for small animal irradiators [159].[167].In addition, it is crucial that appropriate interaction cross-sections and particle energy or distance cutoffs are used and therefore an appropriate application of MC dose calculations requires a good knowledge of the MC code as well as the irradiation system.In order to enable access to MC dose calculations for small animal radiotherapy, an MC dose calculation engine based on an analytical source model and VMC++ was recently implemented in commercial software for small animal treatment planning µ-RayStation [126,168].Full plan dose calculation speeds were quoted in the order of 3-5 minutes, which was up to 680 times faster than full GATE MC dose calculation, rendering µ-RayStation online dose calculations feasible.

Dose calculations
Non-commercial MC dose calculations, while accurate, are still time consuming and might not be feasible for small animal radiotherapy treatment planning tasks.More time-efficient dose calculations are therefore of high importance in small animal radiotherapy.

Other techniques
A number of analytical models that result in faster dose calculations in small animal radiotherapy exist.In general, the analytical approximation is performed in the source description, such as in µ-RayStation described above.Granton and Verhaegen also built an analytical source model of the 225Cx image-guided small animal irradiator and reported a high dose calculation accuracy for field sizes >1 mm and speed ups of a factor of 19-1200 depending on beam size [146].In addition, GPU-  [169].Dose differences between the analytical dose calculations and EBT film measurements and MC of <5% were found for a 5×10 mm 2 beam with ultrafast dose calculation times of < 10 s.In order to fully evaluate analytical dose calculation algorithms, smaller fields sizes of <1 mm should be studied.It is likely that analytical dose calculation algorithms will not be feasible to implement in SFRT employing submillimeter x-ray beams.

Future prospects
In the last decade there has been great progress within the field of small animal radiotherapy including the associated dosimetry.There is a clear need for online dosimetry with energy-independent detectors of sub-millimeter sizes capable of accurate dose measurements of millimeter-size kilovoltage x-ray beams.Small-volume ionization chambers, plastic scintillator detectors, and diamond detectors are currently the most suitable detectors for this task.
In terms of spatially-fractionated radiation therapy, however, no such online dosimeter currently exists.Dose measurements are currently done with radiochromic films that require offline readout and therefore cannot be used for real-time measurements.One future possibility to perform online SFRT dosimetry could be the combination of a scintillating film and a high-resolution CCD camera [169].FLASH radiation therapy is moving into the forefront of small animal radiation therapy research, and as such, suitable detectors need to be characterized for the novel high dose rates and small field sizes of small animal FLASH irradiations [171][172][173].

Conclusions
We provide an overview of small animal radiotherapy dosimetry with a focus on small field and spatially fractionated radiation therapy.Preclinical research is crucial in ultimately making advancements in patient care.We have discussed the challenges with properly measuring the small fields encountered in small animal dosimetry, which is critical in realizing the clinical transition from preclinical research.The main radiation sources that deliver kilovoltage energies in modern small animal studies are presented, including the most optimal dosimeters and phantom materials required to accurately measure kilovoltage x-rays and small field sizes.Current standards and best practices in kilovoltage dosimetry are discussed including the methods used to apply the gold standard in small animal dose calculations.This review will help serve as a compact reference compilation for current standards in small animal dosimetry techniques and provides a foundation for implementing new dosimetry techniques while moving forward within this growing field.

Figure 1 .
Figure 1.A typical dose profile in microbeam therapy (MRT).Important beam parameters are denoted.

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International Conference on 3D and Advanced Dosimetry Journal of Physics: Conference Series 2630 (2023) 012013 , the Code of Practice from the Institution of Physics and Engineering in Medicine and Biology (IPEMB) [139], the International Atomic Energy Agency (IAEA) TRS-398 [140], the Netherlands Commission on Radiation Dosimetry NCS-10 [141], and the most recent German Institute for Standardization DIN 6809-4:2020-04 [142].Dosimetric comparisons of the different protocols have been examined [143-145]

Figure 2 .
Figure 2. Photograph (a) and 3D volume renderings (b, c) of a 3D printed heterogeneous mouse phantom.The plastic scintillator and film dosimeter (1 mm active element in orange, not to scale) sites are outlined in a).Reprinted with permission from Esplen et al[72].

6. 1 .
Monte Carlo simulations Monte Carlo (MC) based dose calculations are gold standard for dose simulation algorithms within the field of clinical radiotherapy as well as for preclinical radiotherapy.A number of MC codes for x-ray beam dose calculations exist, such as the EGSnrc code [160], GEANT4 [161], and its wrapper TOPAS [162], VMC++ [163], MCNPX [164], and FLUKA [165].MC dose calculations with kV x-ray beams 12th International Conference on 3D and Advanced Dosimetry Journal of Physics: Conference Series 2630 (2023) 012013 IOP Publishing doi:10.1088/1742-6596/2630/1/01201310 are highly sensitive to the accuracy of modelling of the incident beam [166] as well as the irradiated object.For example, Bazalova et.al. demonstrated that segmenting at least 39 tissues is required to calculate dose with a 2% accuracy for a 250 kVp beam [116].Muhavava et al presented a MC study that can be used to convert measured dose at a point to dose at a different depth in single-field irradiators

Figure 3 .
Figure 3. Effects of changing geometry on delivered dose in an image-guided small animal irradiator as a function of field size (FS), distance from the source, backscatter, material thickness (T), and attenuation.Adapted from Draeger et al.1 with values specific to conformal small animal irradiators [153, 154, 156].