H7422 40p gaasp photomultiplier tube

All cores in this study were imaged with a custom built forward detection SHG microscope utilized previously [31 , 32 (link)]. A MIRA 900 Ti: Sapphire laser (Coherent, Santa Clara, CA) was used to deliver 780 nm light to the sample using a 40×/1.25NA water immersion objective (Nikon, Melville, NY). Forward channel light was collected using a 1.2 NA condenser (Nikon, Melville, NY) and the SHG signal was filtered with a bandpass filter specific for the collagen signal at 780 nm (390/18 BP, Semrock) and collected with a H7422–40P GaAsP photomultiplier tube (Hamamatsu, Hamamatsu, Japan). Circular polarization was implemented and verified for the SHG light source. All of the cores were imaged with consistent power settings as a montage of 1024 pixel by 1024 pixel image tiles using in house developed acquisition software (http://loci.wisc.edu/software/wiscscan). No SHG signal was observed for five grade 1 cores and four grade 4 cores after navigating the system to at least 3 different fields of view on each core. Hence those 9 cores were excluded. Seventy grade 1 cores and 51 grade 4 cores with SHG signal were imaged and analyzed in this study.

All slides to quantify intratumoral fibrosis collagen density in this study were imaged with a custom-built forward detection of the second harmonic generation (SHG)-based imaging utilized previously [5 (link)]. SHG imaging and quantification of collagen slides were evaluated by SHG Genesis (His-TOIndex Singapore). A MIRA 900 Ti:sapphire laser (Coherent, Santa Clara, CA) was adopted to deliver 780 nm light to the slides using a 40 × /1.25 NA water immersion objective (Nikon, Melville, NY). A 1.2 NA condenser (Nikon, Melville, NY) was used to collect the forward channel light, and the collagen signal was filtered with a bandpass filter at 780 nm (390/18 BP, Semrock) and integrated with a H7422–40P GaAsP photomultiplier tube (Hamamatsu, Hamamatsu, Japan). Then, circular polarization was used to verify the SHG light source. In addition, 100 pieces (including total quantification of collagen and collagen parameters in different regions) of each slide were automatically extracted and analyzed by software to obtain the collagen density.