Goals

(1) to make up in part for the lack of sensory coverage in lecture
(2) to give you a hands-on appreciation of optics and vision
(3) using Drosophila, give you invertebrate animal lab demonstrations
(4) show you some properties of insect vision (notably ultraviolet sensitivity)
(5) demonstrate microscope imaging relevant to rhodopsin's spectral properties
(6) to use a clever iWorx application to measure visual and auditory reaction times


Demonstration

Drosophila phototaxis

A few years ago

Let me attempt to summarize the issue, related to the portion of my research addressed to this topic:

Drosophila have 2 compound eyes that mediate positive attraction to light. Each of about 800 ommatidia (facets) have 8 receptor cells of 3 anatomical types, R1-6, R7 and R8. R7 is an ultraviolet (UV) light receptor that dominates phototaxis at photopic light levels (levels above very dim) even in the rdgB mutant which has selective degeneration of the more sensitive R1-6 cells. It seemed interesting that UV phototaxis was easily demonstrated in rdgB despite the flies' disability. Another mutant, ora= outer rhabdomeres absent, was later shown to be a nonsense allele of the gene for R1-6 rhodopsin. Theoretically, ora should have the same challenge as rdgB, lack of R1-6 function while R7/8 work fine. However, my colleagues and I, testing ora, found several times that it did not have any attraction to light. Thes days, a mutant called ninaEoI17, a big deletion of the R1-6 rhodopsin gene, is used in many studies.

Does ninaEoI17 also lack phototaxis? The 2004 physiology lab class collected pilot data suggesting that they do not. Then, an undergraduate student, Michael Haskins, working in my lab, followed by obtaining the data to prove this finding. His work earned him second place in biology's 2006 undergraduate research award, a monetary award, and recognition in the precommencement program.

This year

This relates to this paper. We will collect pilot data. The stock is "norpA" which results in "no receptor potential" in all 8 types of photoreceptors. The gene function was rescued with a promoter that works in only one type of receptor, R1-6. Do they have phototaxis?

Human UV to blue sensitivity

Where I went to college (Columbia College in New York City), the psychology department was strong in psychophysics. They celebrated "Fechner day," Fechner being the founder of psychophysics (or, somewhat overstated, having solved the mind-body problem). Psychophysics is the study of sensory systems. In this demonstration, we pursue one of my interests, human visual sensitivity to ultraviolet (UV) light. Using the optics in the first paragraph of the hyperlink, we can do what I might call "a poor person's psychophysics." We will compare your UV vs. blue sensitivity and compare it with mine (one eye aphakic and the other eye older than yours). You will also learn about the logarithmic nature of light intensity.

Examine the neutral density filters I put out. The values are in log to the base 10. A 0.3 log unit filter would cut the light in half (you do the arithmetic) [it doesn't seem that much, does it?] and 0.6 would cut it to 1/4. A 0.3 plus a 0.6 (try looking at the room light with the two stacked) is the same as the 0.9. By the way, logs are discussed in your text.

Previous data

How much do you need to attenuate the blue light so it is the same brightness as the UV light?

Here was the determination by the 2005 class. At age 58, I used 4.1 (for my eye that does have a lens). Students and TAs averaged 21.24 in age and used an average of 3.04. Since our goal was to darken the blue light until it appeared the same as the UV, that would imply that the UV looked 1 log unit (10 x) brighter for you than me, consistent with the hypothesis that the lens absorbs more UV light with increasing age.

Exercises

Station 1

Each lab group has a flashlight, but, to economize, no mirror, so work in groups.

#1

Shine the flash light toward one eye while a lab partner looks at the iris of the other eye. Note the pupillary reflex is bilateral.

#2

Have a lab partner look at your iris, identifying landmarks. Roll your head toward a shoulder. Note that, within limits, the eye stays upright, mediated from the vestibular apparatus.

#3

Here is a suggestion from Jeremy Beatty, a former student here in biology and now an Optometrist - the "Purkinje tree." If the room is dark enough or the light source bright enough, have the students shine the light from the side or underneath on the sclera near the iris and shake the light. An alternative that I. as a professor, notice all the time is to walk past the transparency projector in a partially darkened room. The light will cast shadows off of the vasculature that lies over the retina. The result is that the student will see what look almost like tree branches. Under normal conditions these vessels are not seen because of the troxler effect, which is the fading of an object under stabilized conditions.

#4

Determine your dominant eye. Find something in the distance that is just the right size to be hidden from view by your thumb at arm's length. Cover it with your thumb. Close one eye, then the other. Note that your thumb did not cover the object for one eye, the eye that is not dominant.

Demonstrations

On the right is a Mercury arc lamp feeding into a monochromator to make monochromatic lights of the spectrum. In your text, the spectrum is described in Fig. 10-34. We have a monochromator which generates a spectrum using a grating (the obvious alternative being a prism). On the front of the monochromator is a slit that picks off a small section of the spectrum. Using another monochromator, where I could look inside, I obtained this picture to show how the slit selects a portion of the spectrum. The slit picks off 6.4 nm/mm, i.e. if it is 1 mm open, it lets through 6.4 nm of the spectrum.

(1) set the slit at 1 mm
(2) project the beam onto an index card
(3) crank the monochromator and note:

(A) it is brighter at some wavelengths than others

I scanned this graph of the spectral output of various light sources to demonstrate that there are "lines" in the mercury arc (HBO) spectrum at 580, 550, 438, 405 and 365 nm

(B) a setting of about 579 nm looks uniquely yellow

(Just 5 nm higher looks orangish, while just 5 nm lower looks greenish)

(C) at 365 nm, the index card looks blue, but that is fluorescence (roll that round filter that blocks all but UV into the beam because there is white light leak through the monochromator)

(Use glass to reflect the light instead and you will see very little.)

(D) While calibrating the light intensity for the phototaxis research mentioned above, we noted that the light flickered. Many lights probably flicker, but not incandescent lights since the filament stays white hot during alternating current. With different flicker-fusion frequencies, our rods and cones can detect flicker, but not for frequencies as high as alternating current. We can trick our eyse into seeing this flicker by "converting time into space." Jiggling a lens in front of the eye spreads this flicker across the retina, and then the light looks like a dotted line.

Demonstration of Haidinger's brushes

Here is a picture a friend (Lynette Feeney-Burns) gave me before she retired (in about 1990). It is labeled "normal macular pigment - chow diet," and it demonstrates the density of yellow pigment around the fovea in (presumably) monkeys fed a diet adequate in carotenoids. A similar picture is in your book (Fig. 10-28 b), but the darkened area is called the "fovea." Currently, it is known that the carotenoids lutein and zeaxanthin are in nerve layers in the light path to the receptors of the fovea (cones). We get these yellow-appearing caroteinois in our diet (e.g. from spinach and corn). It is thought that they help to protect cones from damage that may be induced by blue light. It was found that the concentration is increased with dietary increases, and now lutein is included in multi-vitamins.

Relevant to this demonstration is that lutein and zeaxanthin are arranged in a way so that they polarize light. It has long been known that insects can utilize patterns of polarized light (for instance, see a book by the 1973 Nobel Prize winner Karl von Frisch, Bees, Ithaca, Cornell University Press, 1950). It is not well-known that polarization is relevant to human vision. Some years ago, polaroid sun glasses were popular because reflected light (glare) had a preferred plane of polarization.

I set out some nice polarizing filters. Handle them carefully. Look through one polarizing filter. Then look through both and rotate one relative to the other, and you will see how they limit light with e-vectors in one plane.

I brought down to the lab room a real nice optical set up. I've set the wavelength at about 460 nm (blue), near the peak absorbance of the macular pigments. I've glued a polarizing filter to a motor that rotates at about 1 rotation per second. Look at the screen. The reason to rotate this filter is that your ability to see the pattern of polarized light is so subtle that you will only notice it if it is continuously changing. What you should see looks like a rotating bow-tie. It is sometimes hard to see, and there are distractions like the light seems to get brighter and dimmer. I found that clear vision is distracting, so it works better for me without my glasses. If you wear glasses, try this. If not, I've set a strong lens nearby that you can use to de-focus your vision.

When I typed "Haidingers brushes" into my search engine, I got lots of fun hits.

There is a huge amount of interest in macular pigments and their possible role in protecting against age-related macular degeneration (ARM). I have several common interests with a St. Louis corporation, ZeaVision. They market nutritional supplements ard are working toward regular clinical measuremtnts of macular pigment optical density as part of a visit to your eye professional.

This relates to the seminar I gave last fall.

Demonstration of rhodopsin - metarhodopsin conversions and green fluorescent protein (GFP)

For this demonstration, groups of students will move down to one of my lab rooms. The techniques are those used by my students and myself in many studies. Rather than write a separate section here, I will hyperlink you to two pages I have put together on this subject. http://starklab.slu.edu/degenvispig.htm
http://starklab.slu.edu/vitArepl.htm

We will
(1) focus a white-eyed fly's pseudopupil on TV and computer with 579 nm light transmitted through
(2) give blue (480 nm) through incident illuminator (same exposure)
What you measured is the decrease in transmission of light near the peak of metarhodopsin in R1-6 receptors induced when blue light converts blue-absorbing rhodopsin to yellow absorbing metarhodopsin. (In flies, visual pigment does not bleach; rather, rhodopsin and metarhodopsin are photointerconvertible).

With a different fly strain, we will inage a photoreceptor marked by green fluorescent protein (GFP).
Using the computer, back in the classroom, I will show you related images from the confocal microscope.

Some of my observations on this line of research are in a recent paper.

Ophthalmoscope

The hard part here is spelling-- o-ph-th... -- one too many h's. The scope has "Feir" scratched into it, so this demo is made possible in part because Dr. Feir, who used to teach this course, bought an ophthalmoscope. It also helped that a close personal friend, Susan Yang, is an ophthalmologist, otherwise I might not know one end of the scope from the other.

Shine it at your hand. Note which dial controls brightness. The lower of the two top dials controls size. Since wie will try to shine most of the light through the pupil that has not been dilated, pick the smallest spot. The top (larger) dial and the switch (left, middle, right) control lens power (diopters). I am strongly myopic (near-sighted, Fig. 10-33), and so my glasses are about -7.5. If everything worked just right (it doesn't), you would have a reading of -7.5 when my retina is in focus if you were examining me without my glasses. Why it does not work just right is that the person doing the examination can change accomodation (Fig. 10-31).

Take turns looking at eachother. Precautions:
(1) keep your fingers and fingernails out of your subject's eyes as you turn the diopter dial when you get close to your subject's eye.
(2) use your right eye if you are examining your subject's right eye (and vice-versa) for social reasons (so you do not bump noses, since you are not Eskimos).

Starting away from the eye, note the red reflex (reflection), like that darn red eye you sometimes get in photos. If you have the subject fixate (point his or her fovea, Fig. 10-36) beyoud your right ear (for right eye examination), you will be examining the nasal retina where the optic nerve exits, see below, blind spot). Move in, changing diopters (watch your fingers!) so that the iris is in focus. If you are luckey, you will see the optic nerve.

Here is how things look in a sheep eye dissection I once had to teach, and you can see the exit of the optic nerve. (That photo I used above to show you the macular pigments is, of course, excellent for seeing the exit of the optic nerve.)

Small field tritanopia

A piece of paper is taped up. Here is what it looks like. Move away from this paper. Look at (point your fovea, Fig. 10-36, toward) the dots. At about the point where the small yellow dot disappears completely (the larger ones are still visible), note that the small blue dot does not look blue (it looks gray). (All red and green dots still look red and green respectively, and the larger blue dots still look blue.)

Explanation. Normally, we have trichromatic color vision, mediated by 3 cone types (Fig. 10-39). Loss of the red mechanism is called protanopia, loss of the green mechanism is called deuteranopia and loss of the blue mechanism is called tritanopia. The human fovea is very low in "blue cones," and thus blue-yellow vision is weak for foveal vision.

(1) Look around the room through the yellow filter I brought and note how clear everything looks even though it cuts out blue input to the retina.

(2) Try to read through the blue filter and notice how hard it is.

Preview of coming attractions

Frequency discrimination and beats

Let's collect some data

We'll use the iWork reaction time program to determine reaction times from all of us and gather some group data.
last year's results
vision average=0.4, standard deviation= 0.088
auditory average=0.2, standard deviation=.051

Last three years' quiz questions

Why is there a blind spot?

there can be no receptors where the optic nerve exits

The macular pigments are in the light path toward the region of sharpest vision called (what)?

fovea

What region of the spectrum are insects famous for being able to see but people cannot?

UV

What is the unit for the power of lenses in contact lenses or glasses?

diopters

How many different cone types mediate normal human color vision?

3

What fraction of the light should get through a 1.2 log unit neutral density filter?

one half to the 4th power = 1/16

A low number of what type of cone is to blame for small field tritanopia.

blue

Suppose a certain amount of light goes through a test tube with water while 1/10 as much light goes through it when a pigment is in the water. What is the absorbance difference?

the log to the base 10 of 10 = 1

You can clearly see a blue appearance by shining 365 nm light onto an index card because of what process?

fluorescence

A myopic patient is examined without glasses by an eye doctor using an ophthalmoscope. (S)he may not get an accurate reading (for the eye glasses prescription needed) because s(he) is subject to the same change of lens shape that compensates for near and far vision. What is this compensatory mechanism called?

accomodation

With the ciliary muscle contracted, the ligaments for the lens slacken. What is this adjustment called?

accomodation

If you had a cataract and I could not test your acuity, how might I test whether your retina still functioned normally? (Hint, we did this.)

you should still see blood vessels with off-axis light

I made the blue light look the same brightness as the UV light with my normal eye (the one with an intact lens) using neutral density filters. I am 58 years old. Predict the relative amount of neutral density for you younger folk. (Alternatively, how did the data collection turn out?)

you will need less n.d. on blue light since your lens is more transparent to UV

It is thought that bees can make use of differences in the plane of polarized light much like we can see colors. Because of how well organized macular pigments are, we see through a polarizing filter. However our polarization sensitivity is so weak that I had to use a trick for you to see polarized light. What was that trick?

kept it changing (rotating)

Why is the wavelength of emitted light longer than that for the excitation light in fluorescence?

energy lost before photon emitted

Drosophila can see UV light. Why can't you?

lens blocks UV

I said that a 0.3 neutral density filter attenuates the light to half. To see if you understand this, you get your calculator and put 10 to the 0.3 power and the answer is 2. Resolve.

attenuation: -0.3 (negative)

Looking straight into an eye with an ophthalmoscope, you would be looking at the fovea. Off to the side is the optic disk. What is the optic disk?

where optic nerve exits (and blood vessels)

Why would it be better for a life raft to be red than yellow?

yellow disappears when it is small

Protanopia is red-blindness. Deuteranopia is green-blindness. What is the term for blue-blindness such as we demonstrated for small foveal visual fields?

small field tritanopia

Why would you use trigonometry in the blind spot test?

with distance from view and distance across, you determine angle off axis

A light is flickering above the flicker-fusion-frequency for your rods and cones. What trick can you use to verify that it is flickering?

look at it through a lens or binocular, jiggle that, change time to space on retina

What are the pigments that contribute to Haidinger's brushes?

macular pigments, zeaxanthin & lutein

What is the name of the part of the thalamus where vision information is relayed?

lateral geniculate nucleus (body)

Give the parameters, roughly, in nanometers, of the visible light spectrum.

400-700

What color light does lutein absorb?

blue

Not everyone saw the bow tie spinning during the presentation of Haidinger's
Brushes. What were we actually seeing?

macular pigments

Name the famous visual pigment associated with rods.

rhodopsin

What is the phenomenon called where Drosophila are attracted to the light?

phototaxis

Blue (480 nm) light was used to do two different things in the
two respective microscope demonstrations. Tell me one of them.

excite fluorescence in GFP (green fluorescent protein
convert visual pigment (rhodopsin to metarhodopsin

The eye adjusts the shape of the lens to keep objects in focus. What is this referred to
as?

accomodation

Are rods or cones more active at night, at low levels of light?

rods

What exits at our blind spot?

optic nerve


This page was last updated 2/6/08

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