Table 1. Commonly used fluorophores and labeling techniques for two-photon imaging
| Probe class | Labeling mechanism | Labeling conditions | Duration and example of use | Representative probes |
|---|---|---|---|---|
| Exogenous | ||||
| Thiol-reactive | Irreversible thiol coupling following esterase cleavage of dye-label ester |
37°C 5-15 μMRPMI or CO2-independent medium (serum-free)30-45 min |
5-7 d Labeling for two-photon imaging |
CMAC CMFDACMTMR |
| Amine-reactive | Irreversible amine coupling following esterase cleavage of dye-label acetate | 37°C 2-8 μMRPMI or CO2-independent medium (serum-free)30-40 min |
5-7 d Labeling for two-photon imagingImaging dividing cell populationsIn vivo labeling of DCs |
CFDA-SE (CFSE)SNARF-1 |
| Loading by membrane- permeant ester | Passive diffusion of small molecule dye into live cells; esterase cleavage renders label membrane-impermeant |
Follow product recommendations | 1-4 h | Fura-2 Indo-1 |
| Active uptake and internalization | Peptide-conjugated probe taken up/internalized by local antigen-presenting cells | s.c. injection of concentrated peptide/probe conjugate (up to 100 μg/imaging site) at the site of imaging or near a draining
LN |
1-3 d Labeling of local antigen-presenting cells or antigen-presenting/collecting cells in a draining LNAntigen distribution tracking |
Antigen-conjugated probe of choice |
| Dye-conjugated dextran | Diffusion, nonspecific blood or lymphatic flow | i.v. or s.c. injection | Tracking blood flow (70-100 kDa, 0.5 mg/100 μL) Tracking lymphatic flow (20 kDa, 0.25 mg/30-50 μL) |
Texas Red, FITC, or rhodamine conjugates |
| QDs | Free or antigen-conjugated | i.v. (20-100 μL) or s.c. (20 pM in 50 μL) injection | Blood flow and perfusion monitoring Induction of an immune response/antigen distribution tracking |
QDs + antigen of choice |
| Carbohydrate binding (whole-tissue labeling) | Sialic acid and lectin-binding dye-conjugated proteins | Soak tissue in 100 μg/mL lectin (prepared in RPMI or CO2-independent medium) for 2-4 h at 4°C |
Same-day labeling and imaging to define tissue structures in vitro | WGA (Alexa 633) |
| Probe conjugated to antibody | Labeling of specific cells in live tissue (presumably by antibody binding to cell surface epitopes) |
Preinjection of 100-200 μg label-conjugated antibody near the draining LN of interest |
Labeling cells and tissues; likely varies with antibody and probe set used (not tested extensively) | Antibody-probe conjugate |
| Endogenous | ||||
| Promoter-driven FP expression | Cell-specific label; all cell types (when FP is expressed under a ubiquitously expressed protein promoter) | Produce transgenic animal or order readily available animal model (some promoters might not induce high enough FP expression to be visible with two-photon imaging) | Lifetime of the cell (label is permanent, nontoxic) Adoptive transfer of purified cellsImaging endogenous cell behaviorProduction of chimeric animals (Witt et al. 2005)Photoconvertible FPs for tracking the behavior ofsingle cells (Tomura et al. 2008) |
Various FPs (e.g., CFP, GFP, YFP) mCherryHcRedKaede |
| SHG | Endogenous highly ordered tissues emit wavelength approximately half of two-photon excitation | Emission best when excitation is perpendicular to structure orientation Excitation at 880-920 nm is preferred for blue/violet channel detection |
Long-term imaging of endogenous structures in various tissues (e.g., LN, skin, lung, muscle) | Myosin, collagen, and tendon |
| Abbreviations: CFDA-SE, carboxyfluorescein diacetate succinimidyl ester; CFP, cyan fluorescent protein; CFSE, carboxyfluorescein succinimidyl ester; CMAC, 7-amino-4-chloromethylcoumarin; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and -6-)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine; DC, dendritic cell; FITC, fluorescein isothiocyanate; FP, fluorescent protein; GFP, green fluorescent protein; i.v., intravenous; LN, lymph node; QDs, quantum dots; s.c., subcutaneous; SHG, second-harmonic generation; SNARF-1, seminaphtharhodafluor -1; WGA, wheat germ agglutinin; YFP, yellow fluorescent protein. | ||||
