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Memo to File

Resumé of discussion with Caspersson, Feb. 14-18, 1961.

A tentative agenda was set up for Tuesday morning, Wednesday afternoon and
Thursday morning to cover the following items:

1) His measurements already conducted of bacterial spectra.

2) Plans for continued collaboration.

3) The design of his instrument.

4) Fundamental measurements that may be required for background,
5) Our technical plans.

6) Organizational problems including suitable staff.

Most of these topics were, in fact, discussed during succeeding conversations
to varying depths. This memo is in draft form and should summarize most of the
items although not necessarily in a completely connected fashion.

1. We discussed our progress in the development of selective filters for the
ultraviolet. He had not been acquainted with cation X nor with the G4S11 lamp.
He was not deeply impressed with the possibility of increasing intensities from
an extended source. However, increases of the order of a factor of 4-4 were
conceded and this might well become quite important for us. Caspersson does not
seem to be too concerned about intensity limitation for his own studies. He
uses primarily a high pressure mercury lamp, Phillips SP500, which he believes
to be essentially equivalent to the GE lamp AH6.

2. Some aspects of his UMSP, the ultramicrospectraphotometer. The high
pressure lamp shows a fall off at the reversion interval but this is mainly
well below 250 nm. His system now uses a Zeiss prism monochromator, |

believe. He has never measured the insertion losses of the optics and at lower
wave lengths, they vary with respect to frequency and also in time. This is one
reason that he does not consider a double path system to be quite satisfactory
and prefers instead to make intermittant recordings of a clear area and of the
sample area. He finds it necessary to make the reference recordings within the
same microscope field. Although he has never made precise measurements, he
would consider that the optical efficiency of his system is at least 25% even
down to 240 nm. In order to reduce glare, the illumination spot should not be
many times larger than the object being studied. In our case this could well
be of the order of 1 micron. The measuring spot in the image plane can be
reduced further to about 0.3 microns. Some of his very elegant spectra are
obtained by direct spectral scanning of a selected spot in the center of the
bacterium. However, for further precision he will often make a zig-zag line
scan ecross the bacterium at each wave length and use the intensity at the
central saddle of the traverse as best representative of the actual absorption.
There is a halo effect at the edges of the bacterium due to the refraction of
the light by the cell. The refractive index of the cell material may be in the
Caspersson, cont. 2

range of 1.42-1.45.

3, We had some discussion of the distorting effects of incomplete obscuration of
the measuring spot by the organism and he feels that it may be important to

allow for mechanical scan in order to optimize the absorption. However, the

main effect of this is not to introduce any spurious qualitative results in the
positions of absorptions but to add a considerable amount of what does amount to
stray light so that the absorption differences will become a smaller percentage
of the total beam.

4, He proposes the, and in fact has used, optical systems for further calibration.

a) The separation of apair of convex and concave lenses for focus and

1

b) The horizontal movement of a thin lens for centering.

5. He believes that we should use a 100:1 projective lens for our measurements.
At the present time we have a 10:1] lens and this may not give adequate magnification.

6. Caspersson was not too optimistic about the possibility of keeping several
objects in focus at the same time for the multiple rapid scan that we had
envisaged. However, this will have to be determined by experience.

{. Caspersson uses polaroid film for routine photography. For more careful
work he uses |lford Photomechanical film.

&. Although in the past Caspersson had expressed some diffidence about this
project, he now indicated that this was mainly because he did not know how
seriously it was being undertaken and in the present circumstances, he is ready
to furnish the most detailed assistance. We therefore had a preliminary
discussion on the kinds of materials that he might fruitfully examine. We
concluded that for the most part, it might be best if we sent him the appropriate
samples. The problem of how to fix the material arose and we considered

that this might be a rather serious problem. However, for the time being,
suspensions in alcohol can be shipped and he can then prepare the necessary
slides. The possibility of handling the material in polyvinyl! alcohol fiims

was also discussed. There was some persistent confusion about this until it

was clarified that this is a water soluble material. Caspersson raised the
point that it should be possible to use oils in place of glycerin as the
immersion fluid and if necessary that Zeiss could make any required re-adjust-
ments In the correction of the lens for the purpose. The present Zeiss lenses
are not homogeneous immersion lenses. If such an oi] can be used, then as we
discussed, it might become practical to have the entire sample holding mechanism
immersed in the ofl. This might simplify the problem of how to conduct the
immersion observation,

9. For more detailed work a suitable precipitant for DNA should be found and
possibly lanthinum in acetic acid may serve this function. tHe was not well
acquainted with the properties of the lanthinum salts in aqueous media and
this should be further studied. His own approach to this problem with tissue
Casoersson, cont, 3

work has been to avoid aqueous media that might be capable of extracting nucleic
acids. He has done considerable calibration work on this and finds that the
absorotivity of his specimens in the ultraviolet does not change over long periods
of time when they are immersed in water-free glycerin; however, small amounts of
water do distort the picture.

10. The Zeiss lenses are calculated for quartz slides of .35 mm thickness.

The same is used as a cover glass. ‘fe have to investigate sources of such slides
for mounting our specimens. Meanwhile, however, Caspersson should obtain these
for us although it might be well to remind him.

ll, We discussed the density separation technique in Ludox which on further
discussion may become not only desirable but essential for the differentiation
of mineral from organic particles. For example, iron oxide which may well have
an embarrassing cutoff at around 2&0 nm should have a density of 5.2. We still
have to determine whether a thin layer of Ludox will cause an inordinate amount
of scattering for the microscopy.

12, Recapitulate on materials to be exchanged: alcohol suspensions of B. subtilis,
E. coli, low density fraction from soil. In addition, some of these samples can

be mounted in PVA, then preparations of particles of ferric oxide and other materials
and other soil components that perhaps should be scanned on a routine basis.

15. Gur agreement to furnish $5,000 towards the stipend for a technician in
Caspersson's laboratory. The detailed way of arranging this will have to be
cleared through HASA and Stanford but should afford no problems.

14, Possibility of replacement of glycerin as an immersion fluid. Caspersson
is looking into this but we should be checking the refractive index onvarious
oils also.

15. Cuestion of source information on mineral absorption spectra. The Landahl-
Bernstein handbook and then there is a tabulation some time ago in Tabulae
Biologiae by Elsevier Press by Ellinger. Caspersson also thinks that Hilger has
a booklet on this. Perhaps this is referred to in some of the other Hilger

books for example, Lothian. (Perhaps see also the University of Michigan summary
on optical materials.

16. Caspersson placed particular stress on the need to localize the optical
axis on the particle and it may be necessary to scan for maximum absorption,
perhaps in the low ultraviolet,

17. The salary stipend would represent about half of Caspersson's costs and he
would like to consider this as a mutual collaboration.

18, We are still somewhat confused as to the present market status of the Zeiss
instruments. According to my notes, Caspersson said that Zeiss was marketing 10
instruments perhaps by the end of this year. In any case, it probably would be
best if either Elliott or | saw the instrument in operation in Stockholm before
making a firm order. On the other hand, it might be well to place a tentative
order in order to be on the priority list.
Caspersson, cont. 4

19. Caspersson's plans are somewhat indefinite. He will be returning to
Stockholm in about a week and he then has a firm date in Geneva on March 25.

He had previously planned to spend 3-41 weeks in Boston after the Geneva meeting.
This would have precluded my own seeing him after Florence. He will inguire in
Boston (Farber) about the possibility of altering his schedule and will write

me by the end of February. Jf he can alter his itinerary, then he could be back
in Stockholm by the 23rd of April. This could match my own plans as | should be
back by the 2th.

20. Fundamental measurements. Caspersson can investigate the light scattering
envelope of bacteria by looking into the effect of the aperture of a field
diaphragm in his microscope on the amount of light collected (a similar procedure
is indicated in a paper by Brackett in the JOSA, 195S). For tissue materials,
the largest part of the ''scattered'' light is found within 20 degrees of the
forward beam. From the shapes of the curves obtained by scanning across a
bacterium, he anticipates that much the same will be true in the ultraviolet for
bacteria and that there should be no real concern as far as the microspectro-~
photometry is concerned. However, the problem of bulk samples of turbid materials
may be much more difficult: 1) im dilute suspension only a fraction of the beam
is attenuated and the signal is a rather weak one superimposed on a rather high
pass 2) With more concentrated suspensions, one must deal with considerable
losses due to multiple scattering before the entire beam is obscured. We

agreed that it would be desirable to measure the scattering envelope of our
specimens as a necessary basis for designing the optical attachments to a
conventional spectrophotometer (in subsequent discugion with the Beckman people,
they agreed to furnish the optical constants so that we will not have to measure
the collecting angle ourselves).

2l. Caspersson brought with him several examples of spectra on individual bacteria.
Most of these cover the range between 250 and 300 or 310 nm. They very
considerably from one specimen to the next, in some cases, absorbancies of as

much as .5 at 260 nm compared to .1 at 300 are seen.

22. Caspersson agreed in principle that he might specifically look for a
technician that could be used on this program in his laboratory and after

perhaps a year's work might be available to come here on loan for continuation

of the program, He does not know whether he will be able to find the appropriate
person,