ATTO 550

Operation Guide (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvents: TE buffer (Tris 10 mM, EDTA (Ethylenediaminetetraacetic acid) (HY-Y0682) 1 mM, pH 8.0); nuclease-free water; 20 mM Tris-HCl buffer containing 50 mM MgCl₂, pH 8.0; PBS.
1.2 ATTO 550-labeled Biotin (HY-B0511) stock solution: prepare at a concentration of 9.23 μM; or prepare at 500 µM in PBS. The excitation/emission wavelengths of ATTO 550-labeled Biotin (HY-B0511) are 554/576 nm.
1.3 ATTO 550-labeled tracrRNA stock solution: resuspend lyophilized CRISPR-Cas9 tracrRNA, ATTO 550 in nuclease-free water to a final stock concentration of 200 µM (200 pmol/µL).
1.4 ATTO 550-labeled gRNA/Cas9 RNP stock solution: incubate negative control crRNA with ATTO 550-labeled tracrRNA at a 2:1 molar ratio at 20 °C for 10 min to form gRNA; complex ATTO 550-labeled gRNA with Cas9 nuclease at a 1:1 molar ratio to form ATTO 550-labeled Cas9 RNP.
1.5 HCR/MOHCR stock solution: prepare 1 µM H1 and 1 µM ATTO 550-H2-BHQ2 in 20 mM Tris-HCl containing 50 mM MgCl₂, pH 8.0. Heat at 95 °C for 5 min, then cool to room temperature.
1.6 Protein/α-synuclein labeling stock solution: label proteins with ATTO-550 using a protein:dye ratio of 1:2 according to the manufacturer's instructions. Use a desalting column to remove unreacted fluorophores. For α-synuclein fibrils, centrifuge at 16,000g for 10 min, resuspend twice in PBS, then label; after labeling, remove unreacted fluorophores by centrifugation at 16,000g for 10 min, and resuspend the pelleted fibrils twice in PBS. Prepare ATTO-550-labeled α-synuclein monomers and fibrils as previously described.
1.7 Store TE buffer stock solution at -20 °C.

2. Working Solution Preparation
2.1 Imaging strands: diluent: PBS buffer containing 500 mM NaCl; for confocal FL-PAINT, use PBS buffer containing 500 mM NaCl. Working concentration: 0.5 nM for each imaging strand in PBS buffer containing 500 mM NaCl; 0.1 nM for each imaging strand in PBS buffer for FL-PAINT; or 0.1 nM for each imaging strand in PBS buffer containing 500 mM NaCl for confocal FL-PAINT.
2.2 ATTO 550-labeled Biotin exposure for screening E. coli: use 0.07 μM to 9 μM ATTO 550-labeled Biotin.
2.3 Cas9 RNP nanoparticles: prepare ATTO 550-labeled NHEJ-NP or CPP-NHEJ-NP from ATTO 550-labeled Cas9 RNP according to the nanoparticle preparation protocol. Working concentration: 6.25 pmol RNP per well.
2.4 Electroporation RNP: prepare sgRNA by mixing 200 µM crRNA and 200 µM ATTO 550-labeled tracrRNA at a 1:1 ratio. Incubate at 95 °C for 5 min using a thermal cycler. Cool at ambient temperature for 15 min under light-protected conditions. Mix 25 µL of 100 µM sgRNA with 20 µL of 40 µM Cas9 to a final RNP complex concentration of 22.2 µM (sgRNA:Cas9 ratio of 2:1). Incubate at room temperature for 10 min. Store protected from light until use. For each sample, transfer 4.5 µL of 22.2 µM RNP complex into a sterile 0.2 mL PCR tube; in a 50 µL total electroporation reaction volume, the final RNP complex concentration is 2 µM.
2.5 α-synuclein assemblies: before cell exposure, buffer-exchange soluble and oligomeric α-syn assemblies into PBS. For in vitro uptake, use 0, 1, 3, or 5 µM ATTO-550-labeled α-syn monomers, oligomers, or fibrils; for Dynasore (HY-15304) experiments, use 1 µM; for in vivo injection, use 1 mg/mL labeled or unlabeled α-syn monomers, oligomers, and fibrils. For cultured neurons, use 1 µM ATTO-550-labeled α-synuclein monomers or fibrils.
2.6 α-SYN assembly labeling: prepare ATTO 550 at a 2-fold molar excess relative to α-SYN assemblies in PBS.
2.7 HCR/MOHCR imaging: diluent: PBS (pH 7.4) containing 0.5% BSA for exosome-bead resuspension. For exosome imaging and cell imaging, use 0.75 µM initiator probe with 1 µM Chol-H1 and 1 µM ATTO 550-H2-BHQ2-Chol. For conventional HCR comparison, use 1 µM H1 and 1 µM ATTO 550-H2-BHQ2 instead of Chol-H1 and ATTO 550-H2-BHQ2-Chol.
2.8 Solution fluorescence assay: mix 0.75 µM initiator probe with 1 µM H1 and 1 µM H2, and incubate at 37 °C for 2.0 h.
2.9 For all working solutions, adjust concentrations as needed and prepare fresh before use.

3. Staining Procedure
3.1 For E. coli displaying Fv-antibody libraries, Biotin-binding Fv-antibodies, intact E. coli, or Fv-antibodies containing only CDR1 and CDR2:
3.1.1 Use Fv-libraries at OD600 = 1.0 for staining and flow cytometry.
3.1.2 Incubate ATTO 550-labeled Biotin (9.23 µM, 1.9 µL) with Fv-libraries (100 µL, OD600 = 1.0) at 4 °C for 1 h.
3.1.3 For concentration-dependent analysis, expose the screened E. coli separately to ATTO 550-labeled Biotin in the range of 0.07 µM to 9 µM or to ATTO 550 alone.
3.1.4 Wash unreacted reagents three times with PBST and PBS.

3.2 For adherent HeLa cells:
3.2.1 Culture in low-glucose DMEM supplemented with 1% Pen/Strep, 1% (w/v) glutamine, and 10% (v/v) fetal bovine serum at 37 °C, 5% CO2.
3.2.2 Seed 16,000 cells onto eight-well chamber coverslips and transfect GFP-PTS1, mito-BFP, and mCherry-FRB-VAPB using transfection reagent.
3.2.3 Twenty-four hours after transfection, wash with PBS, fix with 4% PFA in PBS for 30 min, and permeabilize with 1% Triton X-100 in PBS for 10 min.
3.2.4 Permeabilize and block with PBS containing 2% BSA and 0.1% Triton X-100 at room temperature for 30 min.
3.2.5 Stain with 50 nM nanobody-docking strand conjugate at room temperature with slow orbital shaking for 1 h.
3.2.6 Rinse with PBS, post-fix with 4% PFA at room temperature for 15 min, quench with 0.1 M glycine in PBS, and store in PBS at 4 °C.
3.2.7 Before imaging, rinse the wells twice with 500 µL PBS buffer (pH 8.0, NaCl 500 mM). Place the slide on the microscope stage for 0.5 h for thermal equilibration. Inject the imaging strand mixture containing ATTO 550 at a final concentration of approximately 0.1 nM in PBS, incubate for 5-10 min, then acquire images. For confocal FL-PAINT, inject ATTO 550-labeled imaging strands at a final concentration of 0.1 nM in PBS containing 500 mM NaCl, incubate for 5-10 min, then acquire images. Maintain widefield experiments at 22.0 ± 0.3 °C.
3.2.8 Washing: before adding imaging strands, rinse the wells twice with 500 µL PBS buffer (pH 8.0, NaCl 500 mM).

3.3 For HEK293 cells:
3.3.1 For flow cytometry, seed HEK293 cells in 96-well plates at 10,000 cells per well 24 h before exposure.
3.3.2 For confocal imaging, seed HEK293 cells in 8-well chamber slides at 20,000 cells per well and culture for 24 h before use.
3.3.3 Add ATTO 550-labeled NHEJ-NP or CPP-NHEJ-NP at 6.25 pmol RNP per well and incubate at 37 °C for 4 h for cellular uptake.
3.3.4 For endocytic pathway experiments, preincubate cells with endocytosis inhibitors at 37 °C for 30 min, wash thoroughly three times with 1X DPBS, add ATTO 550-labeled NHEJ-NP or CPP-NHEJ-NP, and incubate at 37 °C for 4 h.
3.3.5 For 4 °C incubation, preincubate cells at 4 °C for 30 min, add ATTO 550-labeled NHEJ-NP or CPP-NHEJ-NP, and incubate at 4 °C for 4 h.
3.3.6 For intracellular trafficking, add ATTO 550-labeled NHEJ-NP at 6.25 pmol RNP per well and assess at 0.5, 2, and 4 h.
3.3.7 After exposure, wash cells three times with 1X DPBS. For intracellular trafficking, after washing, stain with LysoTracker Green DND-26 (75 nM) and Hoechst 33342 (1 µg/mL), then wash three more times with 1X DPBS before imaging.

3.4 For primary mouse OT-I CD8+ T cells:
3.4.1 Use activated cells 24 to 48 hours after activation.
3.4.2 Prepare cells at a final concentration of 1×10⁸ cells/mL for electroporation; standard reactions use 5×10⁶ cells in 50 µL.
3.4.3 Mix 45 µL concentrated cell stock with 5 µL loading agent containing RNP complex (containing ATTO 550-labeled tracrRNA), and mix by gentle pipetting 4 to 5 times.
3.4.4 Perform electroporation using the "Expanded T Cell 3" or "Expanded T Cell 4" protocol.
3.4.5 Store the RNP complex protected from light before electroporation. After electroporation, transfer cells to pre-warmed medium: 3 mL TCM.
3.4.6 Wash cells with electroporation buffer at a volume at least 5 times the pellet volume, centrifuge at 200×g for 5 minutes with low brake, and repeat the wash. After transferring cells, add 50 µL TCM to wash the electroporation assembly wells, pipette 1 to 2 times, and add to the corresponding culture wells.

3.5 For primary mouse BMDM:
3.5.1 Electroporate isolated bone marrow cells, then differentiate them into macrophages for 7 days.
3.5.2 Prepare cells at a final concentration of 1×10⁸ cells/mL for electroporation; standard reactions use 5×10⁶ cells in 50 µL.
3.5.3 Mix 45 µL concentrated cell stock with 5 µL loading agent containing RNP complex (containing ATTO 550-labeled tracrRNA), and mix by gentle pipetting 4 to 5 times.
3.5.4 Perform electroporation using one of the "Opt 0-2", "HSC-4", or "HSC-6" protocols.
3.5.5 Store the RNP complex protected from light before electroporation. After electroporation, transfer cells to pre-warmed Mac differentiation medium.
3.5.6 Wash cells with electroporation buffer at a volume at least 5 times the pellet volume, centrifuge at 200×g for 5 minutes with low brake, and repeat the wash. After transferring cells, add 50 µL Mac differentiation medium to wash the electroporation assembly wells, pipette 1 to 2 times, and add to the corresponding culture wells.

3.6 For primary mouse HSPC:
3.6.1 Expand isolated HSPC for 3 days before electroporation and reseed at 1.0×10⁶ cells/mL in cIMDM.
3.6.2 Prepare cells at a final concentration of 1×10⁸ cells/mL for electroporation; standard reactions use 5×10⁶ cells in 50 µL.
3.6.3 Mix 45 µL concentrated cell stock with 5 µL loading agent containing RNP complex (containing ATTO 550-labeled tracrRNA), and mix by gentle pipetting 4 to 5 times.
3.6.4 Perform electroporation using one of the "Opt 0-2", "HSC-4", or "HSC-6" protocols.
3.6.5 Store the RNP complex protected from light before electroporation. After electroporation, transfer cells to pre-warmed medium: 5 mL cIMDM.
3.6.6 Wash cells with electroporation buffer at a volume at least 5 times the pellet volume, centrifuge at 200×g for 5 minutes with low brake, and repeat the wash. After transferring cells, add 50 µL TCM to wash the electroporation assembly wells, pipette 1 to 2 times, and add to the corresponding culture wells.

3.7 For OLN-93 rat oligodendrocytes:
3.7.1 Culture in DMEM containing Glutamax (4.5 g/L D-glucose), supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator at 37 °C, 5% CO2. Before experiments, differentiate cells under serum-free conditions for 5 days. For confocal analysis, seed onto glass microscope chamber slides.
3.7.2 Expose cells to 0, 1, 3, or 5 µM ATTO-550-labeled α-syn monomers, oligomers, or fibrils for 0, 3, 6, 12, or 24 h.
3.7.3 For dynamin-dependent experiments, expose GFP-expressing OLN-93 cells to 1 µM ATTO-550-α-syn monomers, oligomers, or fibrils in the presence of Dynasore (0, 1, 5, 10, or 20 µM) for 3, 6, 12, and 24 h.
3.7.4 After exposure, remove medium and wash OLN-93 cells three times with PBS. Fix with 4% paraformaldehyde (PFA) at room temperature for 20 min, then wash with PBS to remove residual PFA.

3.8 For mouse cortex in vivo:
3.8.1 Use C57Bl6J male mice and inject into the cortex.
3.8.2 Inject 1 µL of 1 mg/mL labeled or unlabeled α-syn unilaterally into the cortex and analyze 1 h after injection.
3.8.3 For in vivo tissue, perform cardiac perfusion with 0.9% saline, followed by perfusion with 4% PFA in phosphate buffer.

3.9 For α-SYN assemblies in PBS (oligomers, fibrils, or ribbon fibrils):
3.9.1 Label according to the manufacturer's recommendations.
3.9.2 For oligomers, remove unreacted ATTO 550 by size exclusion chromatography; for fibrils and ribbon fibrils, remove unreacted ATTO 550 by three rounds of precipitation and PBS resuspension.

3.10 For MCF-7 cell-derived exosomes:
3.10.1 Adsorb MCF-7 cell-derived exosomes onto latex aldehyde beads before fluorescence imaging. Incubate 10 µL exosomes with 4 µL latex aldehyde beads for 2.0 h. Incubate exosome-beads with blocking solution containing 1 M glycine and 20% BCA (w/v) for 30 min. Wash exosome-beads twice by centrifugation at 5,000 rpm for 3 min, and resuspend in 50 µL PBS containing 0.5% BSA.
3.10.2 Add 0.75 µM initiator probe and incubate at 4 °C for 45 min. Remove free initiator probe by washing three times.
3.10.3 Add 1 µM Chol-H1 and 1 µM ATTO 550-H2-BHQ2-Chol and incubate at 37 °C for 2.0 h.
3.10.4 For photoactivatable exosome imaging, use a blocked initiator probe instead of the initiator probe, then after fuel probe incubation and removal of free probe, expose to 365 nm UV light for 30 min.
3.10.5 Washing: after blocking, wash exosome-beads twice by centrifugation at 5,000 rpm for 3 min; after initiator probe incubation, remove free probe by washing three times; remove free probe before UV exposure.

3.11 For MCF-7 adherent cells:
3.11.1 Seed 5 × 10⁶ MCF-7 cells in glass-bottom dishes and incubate for 24.0 h.
3.11.2 Add 0.75 µM initiator probe and incubate protected from light for 45 min.
3.11.3 Wash three times with PBS.
3.11.4 Add 1 µM Chol-H1 and 1 µM ATTO 550-H2-BHQ2-Chol and incubate for 2.0 h.
3.11.5 Remove free Chol-H1 and ATTO 550-H2-BHQ2-Chol probes by washing three times with PBS.

3.12 For human tonsil B cell subsets (naive B cells and LZ GC B cells):
3.12.1 Place cells on an antigen-containing planar lipid bilayer (PLB) bearing antigen-coupled DNA force sensors.
3.12.2 Antigen-coupled force and control sensors are prepared as previously described. PLBs containing goat F (ab′) 2 anti-human Ig kappa and anti-human Ig lambda as surrogate antigens are prepared as described. Before image acquisition for ratiometric analysis, incubate cells on the antigen-containing PLB for 20 min.

3.13 For streptavidin-coated polystyrene (PS) beads:
3.13.1 Use streptavidin-coated PS beads approximately 20 µm in diameter. For 20 µm PS beads, use a plasma-activated hydrophilic PDMS micropillar array with a width/spacing of 7/7 µm. Pre-wet the micropillar array with 500 nL PBS. Load 1 µL of streptavidin bead PBS suspension containing 8 × 10⁵ beads mL^-1 in four batches. Dry for 2 min.
3.13.2 Perform this procedure at room temperature and 50% relative humidity. Sequentially load 500 nL ATTO 550-labeled Biotin solution onto the four vertices of the square array at 20 s intervals to prevent complete drying. Allow capillary flow to transfer the coating agent through the pillar array; the array can be covered within 5 s. Allow capillary rise to coat the lower half of the beads, forming Janus particles within 2 min.
3.13.3 Washing: before recovery, cut off the four vertices loaded with coating agent to discard unbound coating agent. Load 50 µL PBS and recover the unilaterally coated Janus beads in 1 mL PBS.

3.14 For primary cultured midbrain neurons:
3.14.1 Use primary cultured midbrain neurons derived from wild-type C57BL/6J, D2L-/-, D2 null, or FABP3-/- mouse embryos.
3.14.2 Dissect the midbrain at embryonic day 15.5, incubate with papain at 37 °C for 30 min, mechanically dissociate, and seed at 3×10⁵ cells/cm² onto poly-L-lysine-coated coverslip chambers.
3.14.3 Maintain cultures in Eagle's minimum essential medium supplemented with 10% fetal bovine serum for 1-4 DIV, or in medium supplemented with 10% horse serum for 5-12 DIV, at 37 °C, 5% CO₂ in air, and 100% relative humidity.
3.14.4 Expose cultured neurons to 1 µM ATTO-550-labeled α-synuclein monomers or fibrils for 48 h. For monomer uptake, expose neurons at DIV 10. For fibril uptake, incubate with 1 µM ATTO-550-labeled α-synuclein fibrils for 48 h before fixation.

4. Controls
4.1 E. coli controls: intact E. coli without displayed Fv-antibodies; E. coli displaying Fv-antibodies with CDR3 deleted (or containing only CDR1 and CDR2); ATTO 550 alone instead of ATTO 550-labeled Biotin over the same concentration range.
4.2 Endocytic pathway controls: HEK293 cells exposed to ATTO 550-labeled NHEJ-NP or CPP-NHEJ-NP at 37 °C with or without endocytosis inhibitors, or incubated at 4 °C, to compare cellular uptake.
4.3 Electroporation controls: include buffer control and eGFP mRNA control; include non-electroporated (No EP) cells for comparison with electroporated groups; if a positive control is included, take 50,000 to 100,000 cells for flow cytometry as indicated.
4.4 α-synuclein controls: unlabeled α-syn protein (fibrils or monomers/oligomers) to verify that ATTO-550 labeling does not drive binding to or uptake by cell membranes; Dynasore 0, 1, 5, 10, or 20 µM, with 0 µM as the control for dynamin-dependent uptake; unexposed cultured neurons not exposed to ATTO-550-labeled α-synuclein monomers or fibrils.
4.5 a-SYN assembly controls: free ATTO 550 dye; control animals injected with saline.
4.6 HCR/MOHCR controls: blocked initiator probe without UV irradiation shows no ATTO 550 fluorescence; conventional HCR comparison uses H1 and ATTO 550-H2-BHQ2 instead of Chol-H1 and ATTO 550-H2-BHQ2-Chol.
4.7 Force sensor controls: control sensors lack the hairpin structure and cannot be opened by force.

5. Detection and Analysis
5.1 Instrument types: flow cytometer for FACS analysis and sorting; fluorescence imaging and fluorescence microscopy; confocal laser scanning microscope; two-photon laser confocal microscope; total internal reflection fluorescence microscope (TIRFM); widefield fluorescence lifetime imaging microscope (FLIM) with lifetime camera; time-resolved confocal laser scanning microscope (CLSM) with TCSPC; high-content screening (HCS) reader; in vivo optical imaging system for epifluorescence; ex vivo fluorescence tomography; fluorescence spectrometer; electroporation system for delivery.
5.2 Wavelength settings: ATTO 550 excitation 554 nm, emission 575/576 nm. Flow cytometry FL2 channel excitation 525 nm, emission 572 nm; FL2 gate setting 48-694 A.U., FSC gate setting 104-4200 or 10^4-4200. For HCS, use a three-channel assay with target activation as the analysis application; detect ATTO-550 fluorescence in the red channel with a confocal microscope. For fluorescence assays, excitation 550 nm, emission 560-650 nm; exosome imaging excitation 552 nm, emission 562-700 nm; cell imaging excitation 550 nm, emission 560-700 nm. For fluorescence imaging of ATTO 550-labeled Biotin on beads, use filters with excitation/emission 530-550/>575 nm. ATTO 550-labeled Cas9 RNP is detected in the red fluorescence channel; endosomes/lysosomes are stained with LysoTracker Green DND-26 and detected in the green channel; nuclei are stained with Hoechst 33342 and detected in the blue channel. ATTO 550-labeled tracrRNA is measured in the expected channel for ATTO 550, such as PE or its equivalent channel.
5.3 Result analysis:
5.3.1 E. coli: target E. coli with Biotin-binding Fv-antibodies show significantly higher fluorescence than intact E. coli; mean fluorescence intensity increases with increasing ATTO 550-labeled Biotin from 0.07 μM to 9 μM; over the same concentration range, ATTO 550 alone does not significantly increase mean fluorescence intensity; fluorescence images show the screened E. coli; sort 50,000 cells per sort for target cells.
5.3.2 FL-PAINT: the fluorescence lifetime of ATTO 550 attached to DNA is 3.7 ns; lifetime histograms identify ATTO 550-labeled targets; lifetime thresholds separate targets into individual images; the average localization precision of widefield FL-PAINT images containing ATTO 550 is 17.6 nm; the average localization precision of CLSM FL-PAINT images is 10.8 nm; data analysis can be performed using TrackNTrace/TNT Lifetime Edition software supporting FL-PAINT.
5.3.3 Cas9 RNP: quantify ATTO 550-positive cells by flow cytometry; ATTO 550-labeled Cas9 RNP escapes from endosomes/lysosomes 0.5 h after exposure; at 2 and 4 h, more RNP localizes in the cytoplasm without overlapping with endosomes/lysosomes; at 4 h, considerable overlap with the nucleus indicates nuclear transport; confocal images can be analyzed by Pearson correlation coefficient and Mander's overlap coefficient.
5.3.4 Electroporation: quantify ATTO 550-positive cells by flow cytometry 24 and 48 hours after electroporation; in OT-I T cells electroporated with RNP1 or RNP2, more than 98% of cells are positive; in BMDM, approximately 20% are positive with Opt 0-2, whereas HSC-4 and HSC-6 produce more than 95% positive; in HSPC, approximately 80% are positive at 24 hours with Opt 0-2 or HSC-4, whereas HSC-6 produces approximately 99% positive; higher percentages of ATTO 550-positive cells negatively correlate with target protein optical density on Western blots.
5.3.5 α-synuclein uptake: ATTO-550 signal indicates α-syn uptake; monomeric and oligomeric α-syn are taken up in a time- and concentration-dependent manner, with intracellular ATTO-550 signal visible in CNPase-expressing OLN-93 cells (98% for monomers and 91% for oligomers after exposure to 3 µM for 24 h); fibrillar α-syn rarely enters cells and mainly localizes to the extracellular membrane, colocalizing with β-catenin; after cortical injection, ATTO-550-labeled monomers and oligomers are detected as diffuse or punctate cytoplasmic signal within APC/Olig2-positive oligodendrocytes, whereas fibrils mainly remain around oligodendrocytes; ATTO-550 labeling does not promote binding of α-syn fibrils to cell membranes or uptake of monomeric/oligomeric α-syn.
5.3.6 a-SYN assemblies: analyze 3D reconstruction of ATTO 550-labeled a-SYN fluorescence volumes; fluorescence yield is expressed in pmol M^-1 cm^-1; ATTO 550-labeled a-SYN oligomers diffuse more than fibrils, ribbon fibrils, and free fluorophore; ATTO 550-labeled proteins cannot be imaged in live animals due to high autofluorescence background; confocal analysis detects ATTO 550-labeled human a-SYN deposits in cortical neurons and spinal cord.
5.3.7 HCR/MOHCR: in the absence of initiator, ATTO 550 fluorescence is quenched by BHQ2 and remains low; after initiator activation, ATTO 550 fluorescence recovers and localizes to the exosome membrane or cell contour; MOHCR produces higher ATTO 550 fluorescence than conventional HCR, with an optimal reaction time of 120 min; MOHCR shows a reaction rate 11.6 times faster than conventional HCR, with ATTO 550 fluorescence at 120 min approximately 7.5 times that of conventional HCR; for photoactivatable MOHCR, no fluorescence is observed before UV exposure, and bright ATTO 550 fluorescence appears after UV exposure.
5.3.8 TIRFM force sensor: ATTO 550 is attached to the double-stranded DNA at the top of the sensor as a force-insensitive dye; opening is expressed as the ratio of ATTO 647N signal intensity to ATTO 550 signal intensity; for movies, perform background subtraction and flat-field correction on images in ImageJ, and add a value of 1000 to ATTO 550 images before calculating the ratio; using the threshold function, calculate the ratio of ATTO 647N to ATTO 550 fluorescence in each cell antigen cluster with ImageJ, and calculate sensor opening for each B cell subset using Prism software; time-lapse TIRFM imaging shows that naive B cells rapidly spread on the PLB, capture sensors, and accumulate them at the center of the contact zone within 3 to 4 min, forming a flat immune synapse where sensors persist for several minutes; LZ GC B cells show a punctate aggregation intensity pattern, consistent with BCR binding to sensors concentrated in pod-like structures; strong ATTO 647N intensity coincides with ATTO 550 intensity regions.
5.3.9 Beads: ATTO 550 fluorescence appears green and localizes to the lower half of PS beads, whereas PS bead autofluorescence appears pink; merged images show asymmetrically unilaterally coated Janus beads; no pillar imprints are observed on the bottom of Janus particles; the percentage of fluorophore coverage on PS beads can be controlled up to 48%.
5.3.10 Neurons: quantify ATTO-550 fluorescence intensity in TH+ neurons; uptake of ATTO-550-labeled α-synuclein monomers or fibrils is expected in wild-type TH+ neurons, whereas uptake is reduced in D2L-/-, D2 null, or FABP3-/- neurons; after fibril exposure, ATTO-550 signal can appear as intracellular aggregates in the cell body and as Lewy neurite-like morphology in neuronal processes; assess colocalization of D2L immunoreactivity with α-synuclein fibril ATTO-550 by confocal imaging and Z-dimension analysis.