*2.6. Refractive Index*

For the purpose of verifying the homogeneity of the material and finding a refractive index matching medium for fluorescence microscopy readouts, optical coherence tomography (OCT) was used. The refractive index (RI) measurements of a cured polymer sample were measured using Spectral Domain Optical Coherence Tomography (Telesto-II, Thorlabs Inc., Newton, NJ, USA) by applying the optical path shifting method [28,29] at a wavelength of 1310 nm. At this wavelength, no absorption of light was observed by the polymer material. Only when the optical path length of the sample and reference arm match, the observed interference signal reach a maximum. Hence, it can be used to measure the distance precisely. First, an image of the reflection coming from the scanning stage (glass) was acquired. While keeping the distance between the probe and the scanning stage fixed, the cuboid sample (Type 2) was placed on the scanning stage, and another image was acquired. In OCT measurements, the measured value of the thickness does not present its real thickness but its optical thickness *OT* [29]:

$$OT = n \times T \tag{1}$$

where *n* and *T* are the refractive index and the physical thickness of the sample, respectively. After the sample placement, the position of the scanning stage shifts down due to imaging of material with a refractive index larger than 1, and that optical shift *OS* can be expressed as follows [29]:

$$OS = OT - T \tag{2}$$

Consequently, the refractive index *n* of the sample was calculated using the following equation:

$$n = \frac{OS}{T} + 1\tag{3}$$

The RI of three samples was measured, and the uncertainty of one standard deviation (k = 1) of the result was determined. The physical thickness of the samples was measured using a Millitron Meter (Mahr Feinprüf, Germany, Type 5312340 230 V) with an accuracy of ±1 μm.

#### *2.7. Hardness*

Measurements of the hardness of the polymer material were conducted in order to test if additional post-processing high-temperature heating was needed. The hardness of the polymer material was measured using a Shore A durometer (AD-300, Checkline Europe, Enschede, The Netherlands). Cured, non-irradiated samples (Type 2), as well as samples heated in an oven at 180 ◦C for 10 min, were tested. Six samples, each approximately 1 mm thick, were stacked on top of each other to fulfill the minimal thickness requirement of 6 mm for the measurement [30]. Five measurements of both sample types were carried out, each with a time duration of 15 s.

#### *2.8. Rheology*

Rheological studies of the polymer material were performed in order to determine its viscoelastic properties that contribute to the optimal dosimetric properties. The viscoelastic properties of the polymer material were tested using a rheometer (DHR-1, TA Instruments, New Castle, DE, USA) with a 20-mm parallel plate (Peltier plate steel 106669), a gap of the sample's thickness (1 mm) and temperature of 25 ◦C. Some 1-mm thick cylinder samples were cut out of cuboid samples (Type 2). Amplitude sweep tests were performed for three different samples by applying an angular frequency of 10 rad/s in the strain region from 0.1 to 10% in order to determine the linear viscoelastic region of the polymer material. For the chosen strain of 0.25%, the frequency sweep test of those samples was run in the angular frequency region from 100 to 0.1 rad/s. In addition, a temperature sweep measurement was performed by applying 0.25% strain at an angular frequency of 10 rad/s in the temperature range of 10–100 ◦C. A frequency sweep test was also run for samples irradiated with a low-energy e-beam of energies 80, 120 and 200 keV and a dose of 50 kGy, applying 0.25% strain in the frequency region from 100 to 0.1 rad/s.

#### *2.9. Water Affinity*

The water affinity of the polymer material was tested in order to determine the material swelling properties and the possible use of a water immersion objective for fluorescence imaging. Water contact angle (WCA) measurements were conducted on an OCA20 Contact Angle System (DataPhysics Instruments GmbH, Filderstadt, Germany) using a sessile drop technique at room temperature. A 6 μl droplet of MilliPore water was dispersed through a needle and placed on the surface of the sample. The non-irradiated and irradiated (200 keV, 30 kGy) samples' top, bottom and side surfaces were tested. The WCA was determined using SCA20 software (DataPhysics Instruments GmbH) as an average value of three measurement points on each of the sides.

#### *2.10. Absorbance and Fluorescence Spectra*

The absorbance and fluorescence spectra were measured using a Tecan Spark M10 multimode plate reader (Tecan Trading AG, Männedorf, Switzerland) with a 12-well plate (VWR Tissue Culture Plates). The absorbance spectra were measured as the optical density (OD) in the wavelength range of 300–800 nm with a wavelength step size of 2 nm. The fluorescence (emission) spectra were recorded in the wavelength range of 560–800 nm with a step size of 1 nm, after excitation with 555 nm light of a 5 nm bandwidth. The fluorescence signals were quantified as relative fluorescence units (RFUs) while keeping a constant gain of 100 (the amplification factor for the photomultiplier).

For this, 0.5% *w*/*v* of the leuco-dye was dissolved in the selected solvent (acetonitrile) and divided into two parts: (1) a non-irradiated liquid solution (kept in laboratory conditions up to the measurement time) and (2) an irradiated liquid solution (200 keV e-beam, 30 kGy). The liquid solution was irradiated in a thin (0.5 mm) metal petri dish. The solid samples were prepared as described in Section 2.2 and divided into: (1) a non-irradiated

solid and (2) an irradiated solid (200 keV e-beam, 30 kGy). Additionally, the absorbance and fluorescence spectra of polymer material, irradiated with a low-energy e-beam of 80 keV and 5 doses (15, 20, 30, 40 and 50 kGy), were measured. The spectra were normalized to the absorbance and fluorescence peak value of the 50 kGy sample.

### **3. Results**

#### *3.1. Dosimeter Composition*

#### 3.1.1. Solubility of the Dye

Pararosaniline leuco-dye, used as a radiation-sensitive medium, needs to be fully dissolved before it is mixed with other chemicals. This step is particularly important in order to ensure the uniformity of the samples and their later response to the radiation dose, measured as fluorescence intensity values. Accumulation of undissolved dye might also impair the fluorescence readouts by introducing shadowing or scattering effects. The composition developed by Bernal-Zamorano et al. [19] used ethanol as a solvent. However, our observations indicated that the dye was sparingly soluble, which is also in agreement with the literature values of triphenylmethane dye solubility in ethanol [31]. The dye formed a precipitate and rapidly changed its color once dissolved in ethanol. The dye is reported to be very soluble in solvents like diethyl ether, pyridine, chloroform and benzene [31], but none of these were considered here due to their toxicity. The commercially used Risø B3 radiochromic film [32], which contains the same radiation-sensitive dye as our dosimeter material, uses 2-ethoxy ethanol as a solvent in the fabrication process, and hence it was also tested. Additionally, acetone and acetonitrile were tested due to their lower toxicity and versatility.

All the tested solvents—2-ethoxy ethanol, acetone and acetonitrile—successfully dissolved the required amount of dye. However, the 2-ethoxy ethanol solution very quickly changed its color to magenta and was not further tested, which was also due to its toxicity. Acetone and acetonitrile solutions, after mixing with the rest of the composition and being stored in the refrigerator for 5 days, showed much less coloration. However, the acetone solution started to precipitate after a few days. Due to acetone being more volatile [33] and less polar than acetonitrile [34], acetonitrile was chosen as a solvent for the composition.
