Ti epifluorescence microscope

Manufactured by Nikon

Sourced in Japan

The Ti epifluorescence microscope is a laboratory equipment designed for fluorescence imaging. It provides a core function of illuminating and capturing fluorescent signals from samples, allowing researchers to visualize and analyze cellular and molecular structures and processes.

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Lab products found in correlation

Calcein, by Thermo Fisher Scientific (2 mentions) Ccd camera, by Nikon (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Tsa plus fluorescein, by PerkinElmer (1 mentions) Tsa plus cyanine 3, by PerkinElmer (1 mentions) Tsa plus cyanine 5, by PerkinElmer (1 mentions) Uplansapo 100 oil immersion objective, by Olympus (1 mentions) Imspector imaging software, by Abberior (1 mentions) Ixon ultra emccd camera, by Oxford Instruments (1 mentions) Nis elements ar software, by Nikon (1 mentions) Facsaria 3, by BD (1 mentions) Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)

12 protocols using ti epifluorescence microscope

Standardized Cilia Immunostaining Protocol

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hTERT-RPE1, IMCD3 and MEF cells were grown on coverslips until they reached confluence, and serum-starved for 16–24 h to promote ciliation. Fixation, immunostaining, imaging, and image processing were all as previously reported (Cilleros-Rodriguez et al., 2022 (link)). Briefly, cells were fixed with PBS+4% paraformaldehyde (PFA, 5 min, RT), followed by methanol (3 min, −20°C). For MKS1 staining, only methanol was used. After blocking and permeabilization, primary antibodies were added in blocking solution at the above-indicated dilutions (Reagents and antibodies). After three washes, secondary antibodies were added together with DAPI to stain DNA. Washed coverslips were then mounted and imaged with a Nikon Ti epifluorescence microscope. Images were processed using Adobe Photoshop and/or Fiji/ImageJ. Signal intensity profiles were obtained with the Plot Profile function of Fiji/ImageJ, also as described (Cilleros-Rodriguez et al., 2022 (link)). For HEK293T cells, our protocol was the same, except for two modifications based on recent work: i) coverslips were coated with poly-L-lysine and gelatin; and ii) cells were not serum-starved, as this does not affect their ciliation (Gomez et al., 2022 (link)).

Barbeito P., Martin-Morales R., Palencia-Campos A., Cerrolaza J., Rivas-Santos C., Gallego-Colastra L., Caparros-Martin J.A., Martin-Bravo C., Martin-Hurtado A., Sánchez-Bellver L., Marfany G., Ruiz-Perez V.L, & Garcia-Gonzalo F.R. (2023). EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO. Frontiers in Cell and Developmental Biology, 11, 1190258.

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Neutrophil Adhesion Assay in Microfluidics

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RF24 cells pretreated with control, CD44 or spectrin siRNA were grown in microfluidic chambers of the BioFlux system (Fluxion Biosciences, San Francisco, CA) that had been precoated with 0.05 mg/mL of rat-tail collagen type I in 0.02 M acetic acid. Cells were grown to confluence, washed, and activated with 10 ng/mL of TNF-α for 4 h. Neutrophils were isolated from whole blood as previously described (Riedl et al., 2016 (link)) and labeled with 2.5 μM calcein (Life Technologies). TNF-α was washed away and calcein-labeled neutrophils were introduced to the activated RF24 monolayers at a flow rate of 1 dyne/cm². Images and video were obtained over 10 min using a 4X objective on a Nikon Ti epifluorescence microscope using a Qimaging CCD camera and NIS software.

Mylvaganam S., Riedl M., Vega A., Collins R.F., Jaqaman K., Grinstein S, & Freeman S.A. (2020). Stabilization of Endothelial Receptor Arrays by a Polarized Spectrin Cytoskeleton Facilitates Rolling and Adhesion of Leukocytes. Cell reports, 31(12), 107798.

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Neutrophil Adhesion Assay in Microfluidics

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Quantifying Intracellular Pathogen Activity

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LoVo cells were infected as described above for immunofluorescence experiments with bacteria that constitutively expressed mCherry, as well as eGFP under the control of the P_hlyA promoter. At 1 and 3 h p.i., cells were fixed and stained with Acti-stain 670 phalloidin (Cytoskeleton #PHDN1-A, 70 nM) and DAPI as for immunofluorescence experiments. Fifteen to twenty fields per condition and time point were imaged with a Nikon Ti epifluorescence microscope as described above, and processed with Fiji. Images were first z-projected by maximum intensity, then bacteria were segmented using Otsu’s thresholding algorithm on the mCherry signal. The objects were automatically counted and mapped on the images using the particle analyser. To split grouped bacteria, images were processed with Watershed segmentation algorithm and both mCherry and GFP average signals were measured. For each segmented bacterium, intrabacterial eGFP and mCherry signals were quantified, and intensity of the eGFP reporter of P_hlyA activity was normalized to mCherry intensities. Kruskal-Wallis non-parametric test followed by Dunn’s correction for multiple comparisons was used for statistical testing between conditions.

Peron-Cane C., Fernandez J.C., Leblanc J., Wingertsmann L., Gautier A., Desprat N, & Lebreton A. (2020). Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells. PLoS Pathogens, 16(10), e1009001.

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Multiplex RNA-FISH Analysis of Neuronal Markers

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RNA-FISH was performed using the RNAscope kit (RNAscope® Multiplex Fluorescent Reagent Kit v2; Catalog No. 323100) according to the manufacture’s protocol (ACD-Bio). Target probes used are as follows: RNAscope® Probe- Mm-melanopsin [Opn4] (Cat No. 438061), RNAscope® Probe- Mm- cocaine- and amphetamine-regulated transcript protein [Cartpt]-C2 (Cat No. 432001-C2], RNAscope® Probe- Mm-Cartpt (Cat No. 432001), RNAscope® Probe - Mm-Ecel1-C2 (Cat No. 475331-C2], RNAscope® Probe - Mm-Gm29374–201-C3 (Cat No. 1073601-C3] for lncRNA RP23-416O18.4 and RNAscope® Probe - Mm-TCONS-00067968-C3 (Cat No. 1041891-C3] for lncRNA XLOC_020964. TSA-based fluorophores were from Perkin Elmer (TSA Plus Fluorescein, PN NEL741001KT; TSA Plus Cyanine 3, PN NEL744001KT; TSA Plus Cyanine 5, NEL745001KT). Imaging was done using a Nikon Ti epifluorescence microscope.

Ayupe A.C., Beckedorff F., Levay K., Yon B., Salgueiro Y., Shiekhattar R, & Park K.K. (2021). Identification of long noncoding RNAs in injury-resilient and injury-susceptible mouse retinal ganglion cells. BMC Genomics, 22, 741.

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Visualizing GFP-IRF3 Dynamics in H1299 Cells

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H1299 cells with inducible WTp53 cells stably transfected with GFP-IRF3 were grown onto 1% gelatin pre-coated glass coverslips and after all the treatment cells were washed twice with DPBS and counter stained with Hoechst 33342 and mounted with the ProLong Gold Antifade Reagent. Images were captured with a Nikon Ti epifluorescence microscope and processed using Nikon AR software. For immunofluorescence, cells were grown on glass cover slides and after all the treatment cells were washed twice with DPBS and fixed with 4% paraformaldehyde for 15 mins at room temperature. Cells were then permeabilized with 0.1% triton X-100 and block in 2% BSA for 45 mins. Cells were then incubated with corresponding primary antibodies overnight at 4°C. Cells were washed twice and incubated with secondary antibody for 2 hrs at room temperature. Secondary antibody washed counterstained by DAPI and mount on slides with Fluoromount G. Cells were stained with 3 μl/ml PicoGreen for 1 hr at 37°C and counter stained by Hoechst 33342. Staining was examined under a Leica confocal scanning microscope equipped with a 100× oil-immersion objective.

Ghosh M., Saha S., Li J., Montrose D.C, & Martinez L.A. (2023). p53 engages the cGAS/STING cytosolic DNA sensing pathway for tumor suppression. Molecular cell, 83(2), 266-280.e6.

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Neutrophil and Platelet Adhesion to Endothelial Cells

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Neutrophils and platelets were exposed to BOECs under flow conditions using a Bioflux microfluidic system (Fluxion Biosciences, South San Francisco, CA) as previously described.²² (link) Briefly, the recording channels of 48-well (0–20 dyne/cm²) Bioflux plates were coated with 0.05 mg/ml of rat-tail collagen type I in 0.02 M acetic acid before the introduction of BOECs, which were grown to confluence, washed, and exposed to blocking antibodies for 30 minutes. Calcein-labeled (2.5 μM, Life Technologies) neutrophils were perfused simultaneously with 50% serum (NHS, heat-inactivated serum, C5-depleted serum, C6-depleted serum, patient serum) at a flow rate of 1 dyne/cm², and images and video were obtained after 5 minutes using a ×4 objective on a Nikon Ti epifluorescence microscope using a Qimaging CCD camera and NIS software. Neutrophil adhesion was quantified via total green fluorescence intensity within the measurement channel, obtained via the Bioflux software or counted. For double adhesion experiments, platelets (600 × 10⁶/ml) and neutrophils (12 × 10⁶/ml) in RPMI-1640/HEPES were mixed with NHS at a ratio of 1:1:2, whereas the control channel was loaded with RPMI-1640/HEPES NHS-containing platelets but no neutrophils.

Riedl M., Noone D.G., Khan M.A., Pluthero F.G., Kahr W.H., Palaniyar N, & Licht C. (2016). Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells. Kidney International Reports, 2(1), 66-75.

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Visualizing GFP-IRF3 in Breast Cancer Cells

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GFP-IRF3 positive MDA-MB-231 cells were grown onto 1% gelatin pre-coated glass coverslips and after all the treatment cells were washed twice with DPBS and counter stained with Hoechst 33342 and mounted with the ProLong Gold Antifade Reagent. Images were captured with a Nikon Ti epifluorescence microscope and processed using Nikon AR software. GFP-IRF3 positive H1299 or MDA-MB-231 inducible shp53 cells were grown onto glass cover slides and after all the treatment cells were washed twice with DPBS and fixed with 4% paraformaldehyde for 15 mins at room temperature. Cells were then permeabilize with 0.1% triton X-100 and block in 2% BSA for 45 mins. Cells were then incubated with corresponding primary antibodies overnight at 40C. Cells were washed twice and incubated with secondary antibody for 2 hrs at room temperature. Secondary antibody washed counterstained by DAPI and mount on slides with Fluoromount G.

Ghosh M., Saha S., Bettke J., Nagar R., Parrales A., Iwakuma T., van der Velden A.W, & Martinez L.A. (2021). Mutant p53 suppresses innate immune signaling to promote tumorigenesis. Cancer cell, 39(4), 494-508.e5.

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Fluorescence Imaging with Nikon Ti & Abberior

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Epifluorescent images were taken with an inverted Nikon Ti epifluorescence microscope (Nikon Corporation, Chiyoda, Tokyo, Japan) equipped with a Plan Apochromat 60×, 1.4 NA oil immersion objective, an HBO‐100W Lamp, and an IXON X3897 Andor (Belfast, Northern Ireland, UK) camera, operated via the NIS‐Elements AR software (version 4.20; Nikon).
Confocal images were taken using an Abberior microscope operated with Imspector imaging software (Abberior Instruments, Göttingen, Germany). This setup was built on an Olympus IX83 base, equipped with a UPlanSApo 100× oil immersion objective (Olympus Corporation, Shinjuku, Tokyo, Japan) and an EMCCD iXon Ultra camera (Andor, Belfast, Northern Ireland, UK). Pulsed 561‐nm and 640‐nm lasers were used for excitation of ATTO 590 and STAR 580 and Alexa Fluor 647, respectively. For stimulated depletion, lasers emitting at 595 and 775 nm were employed. Where mentioned, images were deconvolved using Huygens software (Scientific Volume Imaging, WWW.svi.nl).

Yousefi R., Fornasiero E.F., Cyganek L., Montoya J., Jakobs S., Rizzoli S.O., Rehling P, & Pacheu‐Grau D. (2021). Monitoring mitochondrial translation in living cells. EMBO Reports, 22(4), e51635.

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Neuronal and Glial Nuclei Sorting

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Neuronal and glial nuclei sorting were performed as described^{53 (link)} with slight modifications. Briefly, flash frozen mice cortices were homogenized, cross-linked (1% formaldehyde) and quenched (0.125 M glycine) in room temperature for both 5 min. The crude nuclei pellets were further purified through sucrose gradient, stained with conjugated anti-NeuN-Alexa488 (MAB377X, 1:1000) for 20 min and re-suspended into nuclei suspension buffer (0.2% tween-20, 1% BSA, 1x Roche complete EDTA-free protease inhibitor cocktail in 1× PBS). As a negative control, anti-mouse-Alexa488 (A-11029, 1:2000) was used. Sorting was done with BD FACSARIA III containing 85 µm nozzle. Gating was done based on nuclei size, aggregate exclusion and Alexa488 fluorescence. Nuclei were sorted into BSA-coated falcon tubes, spun down, flash-frozen and stored at −80 °C until further processing. The effectiveness of the sorting was confirmed by mounting a small fraction of the sorted nuclei and imaging in an inverted Nikon Ti epifluorescence microscope (Nikon Corporation, Chiyoda, Tokyo, Japan) equipped with a ×20 air objective. Following sorting the nuclei were in-gel digested and processed for MS as explained in the Methods.

Fornasiero E.F., Mandad S., Wildhagen H., Alevra M., Rammner B., Keihani S., Opazo F., Urban I., Ischebeck T., Sakib M.S., Fard M.K., Kirli K., Centeno T.P., Vidal R.O., Rahman R.U., Benito E., Fischer A., Dennerlein S., Rehling P., Feussner I., Bonn S., Simons M., Urlaub H, & Rizzoli S.O. (2018). Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. Nature Communications, 9, 4230.

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