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 |
CMACa 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 imaging;Imaging dividing cell populations;In 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 LN;Antigen 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 cells;Imaging endogenous cell behavior;Production 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 ~ half of two-photon excitation | Emission best when excitation is perpendicular to structure orientation. Excitation at 880-920 nm is preferred forblue/violet channel detection |
Long-term imaging of endogenous structures in various tissues (e.g., LN, skin, lung, muscle) | Myosin, collagen, and tendon |
| aAbbreviations: 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. | ||||
