Visualisering av den immunologiske Synapse av Dual Color Time-gated Stimulert Emission Depletion (STED) Nanoscopy

Summary

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Abstract

Naturlige drepeceller dannes tett regulert, finstemt immunologiske synapser (IS) for å lysere virusinfiserte eller tumorigene celler. Dynamisk aktin omstilling er kritisk for funksjonen av NK-celler og dannelsen av IS. Imaging of F-aktin ved synapse har tradisjonelt benyttet konfokal mikroskopi, men diffraksjon grensen av lys begrenser oppløsning av fluorescensmikroskopi, inklusive konfokal, til ca 200 nm. Nylige fremskritt innen bildeteknologi har muliggjort utvikling av subdiffraction super-oppløsning bildebehandling begrenset. For å visualisere F-aktin-arkitektur ved IS rekapitulere vi NK-celle cytotoksisk synapse ved å følge NK-celler til å aktivere reseptoren på glass. Vi deretter bilde proteiner av interesse å bruke to farger stimulert emisjon uttømming mikroskopi (STED). Dette resulterer i <80 nm oppløsning ved synapse. Heri vi beskrive fremgangsmåten for prøvepreparering og oppkjøpet av bilder ved hjelp av dual coleller STED nanoscopy å visualisere F-aktin på NK IS. Vi belyser også optimalisering av prøven innsamlingen med Leica SP8 programvare og tid-gated STED. Til slutt, vi utnytte Huygens programvare for etterbehandling dekonvolusjon av bilder.

Introduction

Den immunologiske synapse er et komplekst miljø av signalanlegg proteiner og cytoskeletal elementer. Den cytolytiske synapse ble opprinnelig beskrevet som å ha en "blinken" som struktur med en ring av aktin og adhesjonsmolekyler rundt en sentral sekretorisk domene ^1-4. Men nå vet vi at den består av mikroskopiske domener av aktiv signal som krever kontinuerlig dynamisk cytoskeletal omorganisering for funksjon ^5-11. Mye av den informasjonen som vi nå har om synapse er avledet fra mikroskopi, og immunologer har vært tidlige brukere av cutting-edge bildeteknologi.

En slik roman teknologi er super-oppløsning mikroskopi. Konvensjonell lysmikroskopi er romlig begrenset av diffraksjon av lys hindring, som angir den nedre grense for oppløsning for all fluorescens mikroskopi, inklusive konfokal, ved omtrent 200 nm. I de senere årene har flere teknikker vært utvid som tillater oppløsning under diffraksjon barriere. Disse inkluderer stimulering utslipp uttømming mikroskopi (STED), strukturert belysning mikroskopi (SIM), stokastisk løst mikroskopi (STORM), og photoactivatable lysmikroskopi (PALM). Disse teknikkene har blitt gjennomgått i detalj andre steder ^12-15, men er beskrevet nedenfor. Subdiffraction begrenset oppløsning er generert på unike måter i hvert system. Valget av en super-oppløsning teknikk burde derfor være diktert av eksperimentet og eksperimentelt system av interesse.

STED super oppløsning oppnås ved hjelp av en høy intensitet torroidal uttømming stråle som selektivt "stillhet" fluorescens rundt hver Fluoroforen av interesse etter eksitasjon, noe som resulterer i subdiffraction begrenset fluorescens mikros ^16-18. En fordel med STED er at bildet oppkjøpet er rask og krever relativt lite etterbehandling. Mens fargestoff valg er diktert av spectral stilling for uttømming strålen, som i det kommersielt tilgjengelige system ligger ved 592 nm, flere kommersielt tilgjengelige fargestoffer som er tilgjengelige som gjør kombinasjoner av to fluoroforer mulig. I tillegg kan vanligvis brukes fluorescerende reportere som GFP bli fotografert, noe som gjør levende celle eksperimenter mulig ^19,20.

Vi har tidligere brukt STED å identifisere og kvantifisere regioner av F-aktin hypodensity som benyttes av NK-celler for degranulation ^21,22. Vi foreslår at STED er et godt valg for imaging immun synapse på grunn av sin relativt fleksibel tilgjengelighet av fluoroforene og overlegen forbedring i oppløsning i xy-aksen. I tillegg, på den kommersielt tilgjengelige STED system benyttes for disse forsøkene, tillater bruken av en høyhastighets-(12.000 Hz) resonans-scanner for rask anskaffelse av bilder med minimal skade på prøvene. Begrenset fleksibilitet i fargestoff utvalget er ansett som en ulempe av STED ^12,men dobbel farge STED er relativt enkelt med flere kommersielt tilgjengelige fluorophores. Integreringen av STED med en laserskanning confocal mikroskop gir også mulighet for ytterligere confocal bildebehandling i kombinasjon med STED, så mens STED er begrenset til to kanaler, kan flere strukturer avbildes i confocal med oppløsning på ca 200 nm (E. Mace, upubliserte observasjoner ). Mens vi beskriver bruken av STED for avbilding av immunceller, er denne teknologien anvendes på en rekke celletyper, inkludert nerveceller, og for å visualisere en rekke cellekonstruksjoner ^23-26.

SIM bruker en annen tilnærming til å generere subdiffraction begrenset bilder. Ved å visualisere kjente periodisk eksitasjon mønstre, kan informasjonen da bli innhentet om det ukjente strukturen blir studert etter matematisk transformasjon ^27. Dette gir en økning i oppløsningen til ~ 100 nm lateralt ^28,29. Fordelen med SIM er at det jegs kompatibel med alle standard confocal fargestoffer og sonder, men ulempen er at det er mye tregere å hente bilder og disse krever langvarig etterbehandling ^12. Dette begrenser også bruken for live cell imaging.

Endelig kan super-oppløsning bli generert av stokastiske foto-bytting av fluorophores. Denne tilnærmingen er utnyttet i foto aktivert lokalisering mikroskopi (PALM) og stokastisk optisk gjenoppbygging mikroskopi (STORM). Ved å skanne flere kamera rammer og lokalisere tilfeldig aktivert molekyler som er slått "på" og "off" over tid, er bilder med 20-30 nm oppløsning generert fra akkumulert rammer ^30-32. The trade-off for denne resolusjonen er den tiden det tar å hente bilder.

Her viser vi, i detalj, protokollen for å forberede og bildebehandling doble fargeprøver i STED. I dette systemet, er eksitasjon med en pulset, avstembar, hvitt lys, laser. På grunn av naturenav den pulserende eksitasjon bjelke, er tid gating av deteksjon gjort mulig og ytterligere økninger oppløsning. I tillegg er systemet utstyrt med gadolinium hybrid (HYD) detektorer, som er mer følsom enn konvensjonelle fotomultiplikatorrør, noe som tillater en lavere krav til lasereffekt. Uttømming strålen for STED brukes kontinuerlig, og er innstilt på 592 nm, noe som vil diktere valg av fargestoffer som er tilgjengelige for to farger STED. Vanlig brukte fargestoff kombinasjoner inkluderer generelt en nervøs av 488 nm (som Alexa Fluor 488, Oregon Green, DyLights grønne, eller Chromeo 488) og en nervøs av 458 nm (for eksempel Pacific Orange eller Horizon V500). Således, mens deteksjon av de to fargestoffer vil være i et lignende område (og begge er tilgjengelig ved uttømming laser), vil magnetisering oppstå med forskjellige bølgelengder. Med et hvitt lys laser og tunbare detektorer, er å maksimere signal mens eliminere spektral overlapping gjøres ganske enkelt. Som sådan, har vi hatt god suksess med combinationer av kommersielt tilgjengelige fargestoffer, slik som Pacific Orange og Alexa Fluor 488 (benyttet her). Vår protokollen er tilpasset mot, og beskriver evalueringen av humane NK-celler så som representerer historisk fokus i vårt laboratorium. Vi har spesielt anvendelse av NK92 cellelinje i dette eksempel som den som er en vi har ofte anvendt i vårt eksperimentelle arbeid ^21,33.

Protocol

1. Coat Coverslips with Antibody Prewarm (at 37 °C) 30 ml of RPMI 10% FCS media and 1 ml of BD Cytofix/Cytoperm. Prepare a solution of 5 μg/ml of purified antibody in phosphate buffered saline (PBS). For activation of the NK92 cell line, use of anti-CD18  and anti-NKp30 is recommended. Mark one approximately dime sized circle for each condition on a #1.5 coverslip using a PAP pen. For a dual color experiment, there should be four conditions: unstained, dual stained, and two single stained conditions. Dispense 200 µl of antibody solution in each region and incubate at 37 °C for 30 min. Wash coverslips by gently immersing each one in 50 ml of PBS in a 50 ml conical tube at room temperature. Washing should occur immediately prior to the addition of cells and care should be taken to avoid antibody drying on the coverslip. 2. Activate NK Cells on Coverslips; Fix and Permeabilize Isolate 5 x 105 NK92 cells per condition. Centrifuge and decant supernatant. Wash once with 10 ml prewarmed media from step 1.1. Centrifuge and decant supernatant. Resuspend cells in prewarmed media from step 1.1 at a concentration of 2.5 x 106/ml. Gently decant 200 µl to the center of the region created in section 1.2.1. Incubate at 37 °C for 20 min at 5% CO2. (Note: this time can be extended or decreased depending on the biological function of interest. For NK cell granule polarization, 20 min is sufficient). Following incubation of cells, gently wash coverslips by immersing each in 50 ml of room temperature PBS in a 50 ml conical tube. Add 1 µl of Triton X-100 to 1 ml of prewarmed Fix/Perm solution from step 1.1 and vortex thoroughly. Fix and permeabilize by adding 200 µl of Fix/Perm buffer (step 2.3) to cells. Incubate for 10 min in the dark at room temperature. 3. Stain Cells Prepare staining buffer: Phosphate buffered saline (PBS), 1% BSA, 0.1% Saponin. Prepare solution of primary antibody in 200 µl staining buffer (see step 3.1). (Note: antibody should be titrated prior to use). Avoid the use of primary antibody that is raised in the same species used to coat the coverslip (step 1.2). Also avoid Strepatividin-biotin linkages for STED imaging. Following section 2.3.1, gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply antibody solution created in section 3.1.1. Incubate 30 min in the dark at room temperature. (Recommended: incubate coverslips in slide box with a moist paper towel to maintain humidity). Prepare solution of secondary antibody in 200 µl staining buffer. Recommended fluorophores are Alexa Fluor 488, Pacific Orange, and V500. Generally, a 1:200 dilution is suitable for STED imaging. Gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply secondary antibody solution. Incubate 30 min in the dark at room temperature. Repeat washing and staining for additional proteins of interest. If detecting F-actin with Phalloidin, this can be included with secondary antibody, generally at a 1:200 dilution. 4. Mount Coverslips on Slides Prepare mounting medium. Note: Prolong or Prolong Gold are preferable. VECTASHIELD must be avoided, as it is not compatible with STED. 2,2-thiodioethanol must be avoided if Phalloidin is used. Mowiol is acceptable. Place approximately 10-20 µl of mounting medium on a slide. Invert coverslip (cell-side down) and mount coverslip gently, taking care to avoid introduction of air bubbles. Incubate slides for 24 hr (coverslip up) prior to imaging. Seal edges of coverslip with nail polish. 5. Experimental Setup Initiate required lasers and software. Initiate STED depletion laser at 100% power. Align STED laser, which in the case of commercial systems is often an automated procedure. Focus the sample, beginning with single stained control, on the microscope using eyepieces. 6. Optimization of Settings Scan the first channel and optimize laser power, excitation beam position and detector range. If possible, avoid a gain of >100. Capture the image in confocal to optimize settings. Line and/or frame averaging will increase resolution. Check for pixel saturation. Note: Some saturation is acceptable in confocal as application of STED will reduce the intensity of emission. For STED, an optimal pixel size will be below 30 nm however better resolution will be obtained with smaller pixel sizes. The size of the region of interest being imaged will dictate the lower limit of pixel size. Smaller pixel sizes may increase photobleaching. Apply STED depletion beam and capture image, starting with 50% depletion laser power. If an improvement in resolution is seen, more depletion laser power can be applied. At this stage, it may be necessary to adjust excitation laser power, line average, and/or gain. Apply time gating to reduce background (minimum 0.3 nsec). Adjust settings until an improvement in resolution over confocal can be seen. Resolution can be approximated by estimating full width half maximum (FWHM).  This represents the distance at the half maximal intensity of a Gaussian peak created by drawing a line profile across the structure of interest, and is a widely used method of estimating resolution. Once the first channel is satisfactory, initiate a second sequence for sequential scanning. In general, it is best to scan the longer wavelength fluorophore first. Repeat step 6.1 on second channel. Confirm lack of spectral overlap by imaging single stained controls with both scan sequences. Mild spectral overlap can be corrected using spectral un-mixing features in the software, however should be avoided wherever possible. 7. Image Acquisition Acquire images. For quantitative imaging, it is recommended to obtain at least 20 images/condition.  The exact number, however, should be defined according to the experimental question in concert with a statistical approach such as sample size calculation. Save experiment. 8. Deconvolution Open file with deconvolution software or batch processor. Check parameters for each channel using software. Confirm each channel's excitation and emission spectra, STED depletion emission, and imaging direction (up or down, if the image is 3-dimensional) in particular. Deconvolve using the deconvolution wizard. Default settings are generally adequate, however signal to noise ratio (SNTR) will vary from fluorophore to fluorophore and will need to be determined for each channel and each experiment individually.

Representative Results

Clearly, a primary goal of super-resolution imaging will be an improvement over conventional confocal microscopy. However, there are some common pitfalls that may lead to suboptimal resolution. These require that each experiment be optimized individually. In our representative experiment, we are imaging the F-actin network in an NK cell activated by antibody bound to glass. Common causes of (and corrections for) a lack of improved resolution of STED over confocal are as follows: Under-sampling (Figure 1a). This may lead to graininess and loss of pixel information, as shown by poor resolution of F-actin filaments. Increased line or frame averaging can often correct this. Bleaching and/or over-sampling (Figure 1b). This may be caused by lengthy pixel dwell time as a result of excessive line averaging. Alternatively, it may be a result of over-scanning of the image prior to acquisition, including over-use of the depletion laser. This commonly results in hazy or fuzzy images. This can be corrected by scanning the field of interest only minimally before acquiring or, if possible, increasing laser scan speed. If the problem persists, the depletion laser power can be reduced. By achieving the correct balance of pixel dwell time, excitation laser power, and depletion laser power, an image with improved resolution and sufficient information can be generated (Figure 1c). Resolution can be further improved by the use of deconvolution (Figure 1d). When acquisition is optimized, deconvolution will improve resolution both qualitatively and quantitatively and sub-100 nm resolution should be routinely attainable. Figure 1. Optimization of acquisition and common pitfalls of STED imaging. NK92 cells were activated on anti-CD18 and -NKp30 coated glass for 20 min then fixed, permeabilized and stained for F-actin with Phalloidin Alexa Fluor 488. a) An example of loss of image information due to under-sampling. b) An example of loss of resolution due to bleaching/over-sampling c) conditions optimized d) optimized conditions lead to greater improvement in resolution with deconvolution. Scale bar = 5 μm.

Discussion

The improvement in resolution over confocal will be somewhat dependent upon factors which cannot be controlled. These factors include minor aberrations in cover slip thickness and inconsistencies in mounting media. It is important to keep the temperature and humidity in the imaging room as consistent as possible, and the STED beam should be realigned approximately every 60 min. As mentioned in Procedures, use of VECTASHIELD mounting medium must be avoided, as this is not compatible with STED. In addition to which, one should always use #1.5 cover slips, and if available, use those which have been verified to a specific thickness.

One modification of the approach described here is to image additional channels in confocal, using fluorophores that emit at a longer wavelength than the STED beam. In this way, one can image up to four channels (two in confocal, two in STED). If taking this approach, however, the channels with fluorophores emitting above the STED depletion laser will need to be imaged first, as application of the STED beam will deplete photons in these channels. One advantage to this technique is the application of time gating, which will also improve resolution in confocal by eliminating emission from photons with short lifetimes³⁴. In particular, the use of time gating, the timing of emission detectors to correspond with pulsed excitation STED, will decrease background fluorescence from reflection off coverslip glass when imaging close to it. Even in an experiment not suitable for STED, if using a pulsed excitation source, time gating can be a useful tool for improving resolution in confocal.

There are various modifications that can be utilized to improve resolution in STED. One is to decrease the size of the pinhole from the standard 1 Airy unit, although this will also decrease the amount of light reaching the sample. This can be compensated for by increasing laser power or gain. Another is to increase line average, which will increase the amount of information gathered for each photon, improving resolution. Again, however, this may be at the cost of photobleaching of the sample, so a balance will need to be struck between resolution and bleaching. Similarly, use of fluorescent proteins such as GFP will require careful optimization to avoid bleaching. This may be accomplished by decreasing STED laser power if necessary. Longer time points will also allow for greater photon recovery and reduce bleaching. Correction for photobleaching should be accounted for when analyzing live STED.

Of course, imaging in 3 dimensions in STED is also possible, and will also give an improvement over conventional confocal imaging. This is particularly true if it is done in combination with deconvolution, although care should be taken to correct for drift that occurs during imaging multiple planes in the z-axis. If using Huygens software to deconvolve, this correction is obtained using the “stabilize image” feature. Using this approach, resolution in the z-axis will be improved. This is a great improvement over conventional confocal imaging, which has poor axial resolution, and even over just STED itself, which also has relatively poor z-axis resolution. While acquiring multiple stacks in STED, care must be taken to avoid bleaching of the sample, and if necessary one can reduce line averaging or laser power intensity in order to do so. Again, it should be noted that if imaging other fluorophores that are not suitable for STED, application of the depletion beam in the first sequential scan would prevent emission from these channels. Therefore, a mixed STED/confocal approach (when using confocal scanning in channels that emit at a wavelength greater than 592 nm) will unfortunately not be suitable for 3D.

To summarize, we have chosen STED as an approach due to its relative ease of application and improvement in resolution over standard confocal imaging. For imaging the immune synapse, it has proven an effective and valuable technique that allows us to see details in F-actin architecture not possible at resolution over 200 nm. While many of these details seem subtle, they can have a profound effect on NK cell function. Thus, we are applying the latest nanoscopic imaging technology to deriving information that is critical for maintaining human health.

Materials

#1.5 cover slips	VWR	48393-172
BD Cytofix/Cytoperm	BD Biosciences	554722
Bovine serum albumin	Sigma	A2153
Cotton tipped applicator	Fisher Scientific	S450941
Falcon centrifuge tubes (50 ml)	VWR	352070
Fetal calf serum (FCS) (500 mL)	Atlantic Biologicals	S11050
Goat anti-rabbit Pacific Orange	Life Technologies	P31584
Laboratory tissue wipers	VWR	82003-820
Nail polish	VWR	100491-940
NK-92 cells	ATCC	CRL-2407
Phalloidin Alexa Fluor 488	Life Technologies	A12379
Phosphate buffered saline	Life Technologies	14190250
Prolong anti-fade reagent	Life Technologies	P7481
Purified anti-CD18	Biolegend	301202
Purified anti-NKp30	Biolegend	325202
Purified anti-perforin	Biolegend	308102
RPMI 1640 medium (500 mL)	Life Technologies	11875-093
Saponin from Quillaja bark	Sigma	S4521
Super PAP pen	Life Technologies	008899
Triton X-100	Electron Microscopy Sciences	22142
Material Name	Company	Catalogue Number	Comments (optional)
Huygens deconvolution software	SVI	Contact company
Leica SP8 TCS STED microscope	Leica Microsystems	Contact company