Measurement of the mobility of all-trans retinol with Two-Photon Fluorescence Recovery After Photobleaching

Abstract

The mobility of all-trans retinol makes a crucial contribution to the rate of the reactions in which it participates. This is even more so because of its low aqueous solubility, which makes the presence of carrier proteins and the spatial arrangement of cellular membranes especially relevant. In rod photoreceptor outer segments, all-trans retinol is generated after light exposure from the reduction of all-trans retinal that is released from bleached rhodopsin. The mobility of all-trans retinol in rod outer segments was measured with Fluorescence Recovery After Photobleaching (FRAP), using two-photon excitation of its fluorescence. The values of the lateral and axial diffusion coefficients indicate that most of the all-trans retinol in rod outer segments moves unrestricted and without being aided by carriers.

1. Introduction

All-trans retinol is formed in rod photoreceptor outer segments after light excitation from the reduction of all-trans retinal released by photoactivated rhodopsin. It is then transferred to the adjacent pigment epithelial cells where it is converted to retinyl ester by lecithin retinol acyltransferase (1–3). The formation of all-trans retinol removes all-trans retinal, and its transport out of the rod outer segment further improves through mass action the clearance of all-trans retinal. In addition, it feeds the retinoid into a recycling pathway that converts it back to 11-cis retinal used to regenerate rhodopsin. In the rest of the chapter, the terms retinal and retinol without qualification refer to the all-trans isomers. The mobility of retinol makes a significant contribution to the rate of reactions, such as the removal of retinal or the recycling of the chromophore of rhodopsin, in which it participates. Retinol is highly insoluble in aqueous solutions (4) and its transfer across intracellular and extracellular space is typically aided by specialized carrier proteins (5). Thus, in serum, retinol is carried by retinol binding protein (RBP), in the interphotoreceptor matrix by interphotoreceptor retinoid binding protein (IRBP), while within retinal pigment epithelial cells it is transported by cellular retinol binding protein (CRBP-I).

The concentration of retinol can be monitored from its fluorescence (6–9), and its mobility can be measured with Fluorescence Recovery After Photobleaching (FRAP) (10). A FRAP measurement begins with the photobleaching of the fluorophore within a defined volume and then monitors the redistribution of fluorescence as unbleached fluorophore molecules move into that volume. The time course of the recovery of fluorescence reflects the mobility of the fluorophore and can be analyzed to obtain its diffusion coefficient. Today, Laser Scanning Confocal Microscopes usually have all the necessary optical components, including a software module, and can routinely be used for FRAP measurements. Retinol absorbs maximally ~325 nm, which would require the use of an ultraviolet laser line for fluorescence excitation and photobleaching. Another option is two-photon excitation of retinol fluorescence with 700 – 720 nm light (10, 11), and using the same light for photobleaching. Non-Linear Optical Confocal Microscopes incorporate infrared lasers with sufficient power to reach the intensities needed for two-photon excitation of fluorescence (1); they typically have all the necessary hardware and software components to carry out FRAP measurements. An important feature of two-photon excitation and photobleaching is that they take place only at the plane of focus. This has the important advantage of minimizing phototoxicity and fluorophore bleaching out of the focal plane, but it can complicate the analysis of FRAP measurements.

In order to obtain the diffusion coefficient from the time course of the fluorescence recovery of a FRAP experiment, it is necessary to have an analytical expression linking the two. Such an expression is typically obtained by solving the diffusion equation for the movement of the fluorophore. In this study, we have used two-photon fluorescence excitation and photobleaching to examine the mobility of retinol in frog rod photoreceptor outer segments. The cylindrical symmetry of the outer segment simplifies the procedure for solving the diffusion equation and obtaining the requisite analytical expressions (10). A more general approach for the analysis of multiphoton FRP experiments that is independent of the geometry of the system has been presented (12) but requires detailed characterization and knowledge of the bleaching volume.

Because of its low aqueous solubility, virtually all of the retinol in the rod outer segment will be in the membrane compartments, and specifically in the disks, which comprise ~99% of the total membrane. The movement of retinol within the outer segment will therefore consist of diffusion in the plane of the disk membrane and of transfer between the disks. At the cellular level, diffusion in the plane of the disk membrane will be manifested as a lateral movement, perpendicular to the outer segment axis. On the other hand, transfer between the disks will appear as a longitudinal movement, parallel to the outer segment axis. Because of the cylindrical symmetry of the outer segment, the movement of retinol in the lateral and longitudinal dimensions can be measured separately. The lateral diffusion coefficient of retinol measured with multiphoton FRAP was found to be 2.5 ± 0.3 μm² s⁻¹, in close agreement with the diffusion of lipid molecules, suggesting that the bulk of retinol moves freely in the disk membrane. The proper way to measure the lateral diffusion coefficient is through the decline of fluorescence in the unbleached area (Figure 1). Multiphoton FRP measurements also demonstrate that retinol moves along the length of the outer segment, and with a diffusion coefficient of 0.07 ± 0.01 μm² s⁻¹. Because of its limited aqueous solubility, this longitudinal movement of retinol is expected to be via the plasma membrane of the outer segment. This interpretation is consistent with the relative values of the lateral and longitudinal diffusion coefficients and the relative areas of the disk and plasma membranes (10).

Figure 1.

Simulation of the FRAP experiment used for determining lateral diffusion coefficient for a rod outer segment with radius R = 3 μm. The fluorophore (shown as oval-shaped) is assumed to diffuse in the plane of the disk membrane with coefficient D = 2 μm² s⁻¹. The scheme on the right shows a transverse cross-section of the outer segment (bleached areas are lightly shaded and are devoid of fluorophore), with the rod lying on the bottom of the chamber and being scanned from the top. The graph on the left shows the corresponding kinetics of fluorescence recovery in bleached and unbleached regions. Fluorescence recovered in the bleached disk area (●) faster than in the unbleached (△). The lines are single exponential fits with rate constants of 1.1 s⁻¹ and 0.6 s⁻¹ for the bleached and unbleached areas, respectively. Used in conjunction with Eq. 1, these rate constants would result in apparent diffusion coefficients of 4 μm² s⁻¹ and 2.2 μm² s⁻¹. Used with Eq. 1, the rate of fluorescence decline in the unbleached area provides a good estimate of the lateral diffusion coefficient. Reprinted from (10) with permission.

2. Materials

A dark room is necessary for dark-adapting animals and for dissection. An area of ~50 sq.ft. is sufficient. A revolving door for entering is convenient, but a thick black curtain is also adequate.

2.1 Photoreceptor cell preparation

1. Red lights for the dark room. They are obtained from photographic equipment stores, nowadays through the internet. One choice is adjustable Kodak safelights with filters number 2. For individual red bulbs, an appropriate choice is the Delta 1 Jr. Safelight. It is best to keep the red lights as dim as possible.

2. Frogs (Rana pipiens) are obtained from approved vendors (The Sullivan Company, Nashville, TN; NASCO, Fort Atkinson, WI and Modesto, CA; or Carolina Biological, Burlington, NC). Check with the supplier well in advance for seasonal availability.

3. Amphibian Ringer’s with composition: 110 mM NaCl, 2.5 mM KCl, 1.6 mM MgCl₂, 1 mM CaCl₂, 5 mM HEPES, pH = 7.55. The pH should be adjusted to the final value with NaOH. The Ringer’s solution can be kept well-sealed at room temperature for months [see Note 1].

4. Stock glucose solution, 1 M. This solution has to be kept at −20 °C to avoid bacterial growth.

5. Dissecting microscope.

6. Infrared light source. It can be a home-made infrared-LED-based system. Alternatively, an infrared safelight (FJW Optical Systems, Inc.) can be used.

7. Two infrared image viewers attached to the dissecting microscope eyepieces. The viewers are the FIND-R-SCOPE Infrared Viewer Model 84499A (FJW Optical Systems, Inc.). The same company also provides the components necessary for attaching the viewers to the eyepieces.

8. An infrared viewer with illuminator. An option is FIND-R-SCOPE Infrared Viewer with Illuminator Model 85100A (FJW Optical Systems, Inc.).

9. Petri dishes. 35 mm Falcon (Fisher Scientific).

10. Plastic transfer pipettes, 5 mL (Fisher Scientific).

11. Dissecting tools (Fine Science Tools or Roboz Surgical Instruments). One pair of delicate iris scissors, straight, 11.5 cm long. One pair of extra fine Bonn scissors, curved, 8.5 cm long. At least two pairs of fine Dumont forceps, numbers 5 or 7. A couple of pairs of inexpensive student Dumont forceps (number 5) are also useful during the dissection. One blade holder/breaker. Pithing needles.

12. Sylgard 184 elastomer kit (Essex, Charlotte, NC).

13. Metal cutter (local hardware store or Fisher Scientific).

14. Double-edge razor blades, “Personna Double Edge Platinum Chrome” (Wal-Mart).

15. Experimental chambers. These can be 35 mm culture dishes with a 12 mm chamber (Warner Instruments, Hamden, CT).

16. Coating solution for chambers: 0.01% solution of Poly-L-Ornithine or 0.1% solution of Poly-L-Lysine (Sigma-Aldrich Chemical Company, St. Louis, MO). The Poly-L-Lysine solution is diluted with distilled water to a final 0.01% concentration.

17. Three light-tight boxes that can accommodate 2–3 of 35 mm Petri dishes each.

18. Fiber optic illuminator and longpass (>530 nm) filter for bleaching the cells (Edmund Optics, Barrington, NJ).

2.2 FRAP measurement

1. Non-Linear Optical Confocal Microscope with a Ti-Sapphire tunable infrared laser. The software running the system typically includes a program for setting up the parameters for the FRAP experiment. There are several such systems available, for example the one based on the Zeiss LSM 510 (Carl Zeiss, Thornwood, NY). Make sure that the experimental chambers fit on the microscope stage. A special stage accessory that is usually available from the microscope manufacturer might be necessary.

2. High numerical aperture objective lens. For a system based on an upright microscope, use the 63× water immersion lens (NA = 0.9). For a system with an inverted microscope, you could use either the 40× or the 63× oil immersion lenses.

3. Methods

3.1 Photoreceptor cell preparation

3.1.1 Dishes, chambers, and razor blades

1. Coat the bottoms of 35 mm Falcon Petri dishes with Sylgard elastomer. Prepare the elastomer according to the instructions on the box and pour a small amount in each dish to cover its bottom with a thick layer. Replace the covers on the dishes and store them. The elastomer will harden over a period of a few days and the dishes will be ready.

2. Coat the bottoms of the experimental chambers with 0.01% poly-L-lysine or poly-L-ornithine; 200 μL of solution per chamber is enough. Cover the chambers with a paper towel to protect them from dust and let them sit until dry. Wash them with distilled water and keep them upside-down to dry. Store in a closed box and use within 2 weeks.

3. The chambers can be re-used. After an experiment, wash the chamber with 100% ethanol to remove oil on the outside (from the oil-immersion lens) and the cell debris on the inside. Use cotton-tipped applicators to gently scrub the bottom of the chamber to remove the debris. Wash with distilled water and let dry.

4. Prepare several small razor blades by cutting each double-edged blade into 8 pieces with the metal cutter.

3.1.2 Isolated retinas

1. Keep the animals healthy and clean, feed them and provide veterinary care [see Note 2].

2. Dark-adapt an animal in a ventilated container (for example, a suitably modified bucket) in the dark-room for at least 2–3 hours before beginning experiments.

3. Immediately before the experiment, add 0.5 mL of the glucose stock to 100 mL of the Ringer’s (final glucose concentration of 5 mM). Use this Ringer’s for experiments. Discard the leftover solution at the end of the day, as it might grow bacteria.

4. Pour some of the Ringer’s solution to two 35 mm Petri dishes and keep them close to the dissecting microscope.

5. Sacrifice the animal under dim red light by pithing the brain and the spinal cord with the pithing needles.

6. Enucleate the eyes using the long scissors and the student Dumont forceps.

7. The rest of the procedures are carried out under the dissecting microscope using infrared light. Use the infrared viewer with illuminator, in case you need to find something outside the field of view of the microscope.

8. Remove any leftover muscle and skin tissue using the long scissors and the student Dumont forceps.

9. Remove the anterior part of the eye, leaving the vitreous behind: use the short scissors to make an incision and cut around just behind the ora serrata.

10. Transfer the eyecup into one of the Petri dishes filled with Ringer’s. Carefully remove the vitreous using the fine forceps.

11. Under the infrared light, the retina is now visible against the dark background of the retinal pigment epithelium. Gently separate the retina from the epithelium; it will remain attached to the eyecup at the optic nerve. With the fine forceps reach underneath the retina and pinch it off at the point of attachment. Separate the retina fully from the eyecup [see Note 3].

12. Using a plastic pipette, draw some solution containing the retina and transfer it to the other, clean Petri dish. It can be kept there in a light-tight box for a few hours [see Note 4].

3.1.3 Isolated living photoreceptor cells

1. Bring pipettes, coated chambers, sylgard-covered dishes close to the dissecting microscope. Grab a piece of razor blade with the blade holder, with the edge of the blade at approximately 45° angle to the holder. All subsequent procedures are carried out under the dissecting microscope using infrared light.

2. Using the small scissors, cut the retina into four pieces. With a plastic pipette, draw some solution containing a piece and transfer it into a sylgard-covered dish. The final volume of the solution in that dish should be about 600 μL.

3. With the fine forceps flatten the piece of retina on the sylgard surface, keeping the photoreceptor side up. Using the razor blade, chop the piece in one direction; repeat 3–4 times, then rotate the dish 90° and chop again 3–4 times. Repeat the whole procedure until you see a “cloud” of dissociated cells. It is important to chop finely, while keeping the piece of retina stuck to the sylgard. If the chopping is too coarse, or the retina becomes unstuck, one gets mostly pieces of retina instead of isolated cells [see Note 5].

4. After finishing the chopping, transfer the solution to 3 experimental chambers, 200 μL in each chamber [see Note 6]. Keep the chambers with the isolated cells in a light-tight box.

5. Wait for 10 min for the cells to settle, then add 2–3 mL of Ringer’s to each of the dishes that contain the chambers. The cells can now be taken to the Non-Linear Optical Confocal Microscope for experiments. Isolated cells can survive for a few hours [see Note 7].

3.2 FRAP measurements

1. Tune the Ti:Sapphire laser to 720 nm for fluorescence excitation. Set the fluorescence emission measurement from 400 to 650 nm.

2. Take one of the chambers containing cells out of the light–tight box, and expose the cells to >530 nm light for 1 min, using the illuminator and the longpass filter. Carry out measurements between 1 and 2 hours after bleaching.

3. Find a cell under the bright field. Only rod outer segments with attached ellipsoids can generate retinol. Among those, it is best to use whole intact cells (with outer segment, ellipsoid, and nucleus) instead of ROS-RIS, as the latter might not survive through the full course of a FRAP experiment [see Note 8].

4. Carry out preliminary measurements to optimize the measuring and bleaching intensities. Along with the intensities, you need to optimize the number of time points, time delays between scans, and the time for bleaching. For FRAP experiments, a high intensity of the laser beam is used to bleach retinol, and a lower, non-bleaching intensity is used for scanning and measuring the fluorescence before and after bleaching. Select intensities and number of scans according to the following criteria: (a) avoid bleaching of fluorescence during scanning, (b) avoid visible cell damage or death (due to phototoxicity), and (c) obtain measurements of sufficient resolution to determine the kinetics of retinol fluorescence recovery [see Note 9].

3.2.1 Measurement of lateral diffusion

1. Select an intact rod cell. Ensure that the whole cell is included in the scanning area, but keep the area small to minimize the time required for frame acquisition. Set up the FRAP parameters and select the area for bleaching. This area should be a rectangular area, covering one half of the outer segment, on one side of the long axis (Figure 2). It is critical that the inside edge of the area is the long axis of the outer segment.

2. Carry out the experiment. The fluorescence should redistribute and equilibrate between the bleached and unbleached halves over a period of 10–20 sec.

3. For each time point after the bleach, measure the fluorescence in a region of interest that covers the unbleached half of the outer segment – opposite to the bleached side.

4. The fluorescence of retinol declines in this area after bleaching. Fit this decline with a single exponential function (for example with a graphics software program like Kaleidagraph), and obtain the rate of decline, k (Figure 2G).

5. D_lateral , the coefficient for diffusion of retinol in the plane of the disk, is given by:

   $$D_{\mathit{lateral}}=d^2\times \frac{k}{{\pi }^2}$$ (1)

   where d is the diameter of the outer segment. For the cell in Figure 2, d = 7.2 μm, and k = 0. 25 sec⁻¹, giving D_lateral = 1.3 μm² sec⁻¹ (10).

Figure 2.

Measurement of the lateral mobility of all-trans retinol in the outer segment of an isolated intact frog rod with two-photon FRAP. (A) Diagram of the cell: ROS, rod outer segment; ell, ellipsoid; the bleached area is shaded. (B) Initial image acquired before retinol bleaching, (C-E) images acquired immediately, 2285 ms, and 6092 ms after bleaching, respectively. (F) Fluorescence profiles along an outer segment diameter in the bleached region from images B (curve 1), C (curve 2), D (curve 3), and E (curve 4). (G) Kinetics of fluorescence recovery in the bleached (●) and unbleached disk areas (△). The lines represent single exponential fits, with rate constants of 0.75 s⁻¹ for the bleached area, and 0.25 s⁻¹ for the unbleached area, respectively. The fluorescence recovery in the bleached disk areas is due to retinol movement from the unbleached areas above and below the plane of bleaching, as well as to retinol movement from the areas in the left side. Images B-E are shown at the same intensity scaling. Scale bar is 10 μm. Reprinted from (10) with permission.

3.2.2 Measurement of axial diffusion

1. Select an intact rod cell. Set up the FRAP parameters and select the area for bleaching. This area should be a rectangular area, covering the top half of the outer segment (Figure 3). It is critical that the inside edge of the area is at the half-point between the base and tip of the outer segment.

2. Carry out the experiment. For long outer segments, it is unlikely that the fluorescence will equilibrate fully between the bleached and unbleached halves within a reasonable time.

3. Measure the values of fluorescence in the unbleached (F1) and bleached (F2) halves immediately after bleaching. Calculate Δ F = F1 − F2. For the cell in Figure 3, ΔF = 50

4. At a time point 20–30 min after the bleach, measure and plot the profile of fluorescence along the length of the outer segment. Measure the slope of the fluorescence, S, at the middle of the outer segment (Figure 3F). For the cell in Figure 3, S = −2.4 μm⁻¹.

5. Obtain the rate parameter α by solving the equation (for example with Mathcad):

   $$S=-\frac{2\times \mathrm{\Delta }F}{L}·\sum _{m=0}^{\infty }{e}^{-{(2m+1)}^2\alpha t}$$ (2)

   where L is the length of the outer segment.

6. D_axial , the coefficient for diffusion of retinol along the length of the outer segment, is given by:

   $$D_{\mathit{axial}}=L^2\times \frac{\alpha }{{\pi }^2}$$ (3)

   where L is the length of the outer segment. For the cell in Figure 3, L = 57 μm, and a = 6.9 × 10⁻⁵ sec⁻¹, giving D_axial = 0.023 μm² sec⁻¹ (10).

Figure 3.

Measurement of the axial mobility of all-trans retinol in the outer segment of an intact frog rod. (A) Diagram of the cell: ROS, rod outer segment; ell, ellipsoid; the bleached area is shaded. (B) Initial image acquired before retinol bleaching, (C-D) images acquired immediately, and 1440 s after bleaching. (E) Fluorescence intensity profiles before (thin line) and immediately after (thick line) bleaching. (F) Fluorescence intensity profile 1440 s after bleaching; the straight dashed line represents the slope of the intensity profile at the boundary between bleached and unbleached regions. The slope of the intensity profile, S = −2.4 μm⁻¹ gave a rate constant α = 6.9 × 10⁻⁵ s⁻¹. Images B-D are shown at the same intensity scaling. The fluorescence intensity profiles are aligned with the images. Reprinted from (10) with permission.