Plan apochromat 20 objective

The imaging system was built on a commercially laser scanning microscope platform (LSM 880 Zeiss, Germany) using a mode-locked femtosecond Ti:Sapphire laser that emitted linearly polarized 810 nm excitation light. The backscattered signals from tissue samples were simultaneously obtained via two independent channels. One channel detected second harmonic generation (SHG) signal (green color) between 395 nm and 415 nm while the other channel detected two-photon excitation fluorescence (TPEF) signal (red color) between 428 nm and 695 nm. A Plan-Apochromat ×20 objective (NA = 0.8, Zeiss, Germany) was employed to acquire large field images. Larger scale imaging was enabled by a motorized stage under computer control (ZEN 2.3 SP1 software). The lateral resolution was ~0.8 µm while the imaging field of view was about 0.5×0.5 mm², typically.

For all fluorescence microscopy analyses, 5 × 10⁴ cells were plated on a 12 mm-diameter glass coverslip. CMs were fixed with 4% PFA and stained with cardiac Troponin T (cTnT, 1:2000, ab64623, Abcam, Cambridge, MA, USA) and JPH2 (Junctophilin-2, 1:200, sc-377086, Santa Cruz Biotechnology, Dallas, TX, USA) primary antibodies, followed by labeling with secondary antibodies. Staining of nuclei was achieved with Vectashield antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained with a Zeiss Axio Scope.A1 upright fluorescence microscope with a Plan-Apochromat 20× objective with a numerical aperture of 0.8 and Zen 2010 software (Carl Zeiss, Frankfurt am Main, Germany). In certain images, the brightness was changed linearly with an open source image processing package Fiji (1.52n), that is based on ImageJ (NIH, Bethesda, MD, USA) [32 (link)].

All the specimens were sectioned into two slices with 5 μm thickness. One was to locate all TN with H&E staining, and another deparaffinized and unstained section was to acquire MPM images and identify the classification of TN. The imaging system was built on a commercial laser-scanning microscope (LSM 880 Zeiss, Germany) using a mode-locked femtosecond Ti:sapphire laser (Chameleon Ultra, Coherent, USA) that tunable from 680 to 1080 nm operating at 810 nm [29 (link)]. The backscattered signals from tissue samples were simultaneously obtained via two independent channels. One channel detected an SHG signal (green color) between 395 and 415 nm, while the other channel detected a TPEF signal (red color) between 428 and 695 nm. MPM images were obtained by scanning unstained histological sections in the full field of vision by using a Plan-Apochromat × 10 objective (NA = 0.45, Zeiss, Germany) to determine the location of TN [29 (link)]. Areas with significantly abnormally strong TPEF signals and containing at least 10–15 adjacent, dead tumor cells were identified as the presence of TN [13 (link)]. Areas, where uncertainty existed as to whether the TPEF signal was enhanced, were coded as the absence of TN. Subsequently, the region of interest was amplified by using a Plan-Apochromat × 20 objective (NA = 0.8, Zeiss, Germany) to obtain the MPM images of TN (Fig. 1C).

MPM image acquisition was achieved using a previously described nonlinear optical imaging system [15 (link)]. Briefly, a commercial laser scanning microscope (LSM 880, Zeiss, Germany) equipped with a mode-locked femtosecond titanium (Ti): Sapphire laser (Chameleon Ultra, Coherent, USA) was used to obtain high-resolution images. The excitation wavelength (λex) used in this study was 810 nm. The backscattered signals were obtained via two independent channels at the same time: one channel for detecting SHG signal (green color) was set between 395 and 415 nm, the other channel for detecting TPEF signal (red color) was set between 428 and 677 nm. A Plan-Apochromat 20 × objective (NA = 0.8, Zeiss, Germany) and Plan-Apochromat 63 × oil objective (NA = 1.4, Zeiss, Germany) were employed for acquiring images from tissue samples. For the purpose of confirmation, a comparison of the MPM image to the picrosirius red-stained serial tissue slice was performed by an experienced pathologist.

For visualization, GUVs were collected
after the electroformation procedure and transferred to an imaging
chamber containing a glass slide previously functionalized with 1
mg/mL bovine serum albumin. The ionic composition of the external
solutions used for these experiments was 100 mM NaCl, 10 mM CaCl₂, 20 mM Tris-HCl and 85 mM glucose (pH 7.4, osmolarity ∼
360 mOsmol/L). For control experiments, CaCl₂ was replaced
with an equimolar osmotic concentration of glucose. Confocal Microscopy
was performed with a Zeiss LSM 880 confocal microscope using a Plan-Apochromat
20× objective. The excitation/emission profile for each of the
fluorophores used was as follows: 2-NBDG or NBD-PE was excited with
a 458 nm Argon laser and emission collected at 525 nm, Rhod-PE was
excited with a 561 nm diode-pumped solid-state laser and emission
collected at 583 nm, and CY5 was excited with a 633 nm HeNe laser
and emission collected at 664 nm. Image processing was performed using
ZEN 3.3 (blue edition) and ImageJ.

We used PTEN and ETS transcription factor ERG IHC data from previous work (24 (link)). TMA slides were costained with anti-FAP (ab207178 rabbit 1:1,300 dilution) plus anti-SMA (M0851, mouse 1:500 dilution) using anti-rabbit (DPVR55HRP, Bright Vision) and anti-mouse (DPVM55AP, Bright Vision) secondary antibodies and the following substrates: Bright Vision, Bright DAB BS04–110 and Liquid Permanent Red K0640, Dako. The slides were washed for 1 minute in water after each reaction. Slides were then counterstained with hematoxylin (1:10 water dilution, 30 seconds) and after water washing and air drying, the slides were mounted (Pertex).
The stained slides were imaged with Pannoramic 250 Flash III (3DHISTEC Ltd) using 20× Zeiss Plan-Apochromat 20× objective (NA 0.8; 0.25 µm/pixel). Images were exported as whole-slide TIFFs and TMA spots were cropped with FIJI Roi1 1-Click tool.
We used Ilastik-1.3.3 machine learning software for FAP and SMA pixel detection using the pixel classification tool. Here, all the pixels were classified either to empty (no stain), FAP-positive, SMA positive, or other tissue (hematoxylin positive + tissue background). Simple segments were exported as TIFFs and classified pixels were counted using CellProfiler. FAP- and SMA-positive pixel counts were then normalized with total tissue pixel counts.