Double Emulsion Autoradiography for Identifying Tritium-Labelled cells in sections
The identification of tritium-labeled cells in tissue sections by the conventional autoradiography methods gives high resolution insofar as localization of T is concerned; however, their sensitivity for measuring the radioactivity is low, and only about 0.5 to 2% of the BETA particles that are emitted from the tissue section during exposure are recorded. Thus large doses of T have to be used. Another disadvantage is that the combination of the low range of T BETA particles and the thickness (4 to 5 mu ) of ordinary tissue sections enables some cells which contuin the isotope to be visible microscopically but to be such a distance from the emulsion as to be invisible autoradiographically. Thus only a proportion of the labeled cells will be identified. The method described lessens some of these difficulties by using the incorporation of thin tissue sections between 2 layers of photographic emulsion. Thus, the sensitivity of tissue autoradiography can be doubled and the number of false-negative cells nearly eliminated. To demonstrate this, a mouse was given 50 mu C of tritiated thymidine intraperitoneally and killed 1.5 hr later. A portion of the small bowel was removed, fixed, and embedded in methacrylate in the usual way. Sections 2 mu thick were cut and allowed to flatten on water at 40 deg C. Some sections were used to make conventional single emulsion autoradiographs and other sections were interposed between 2 layers of emulsion by first coating slides with NTB 3 emulsion, picking up the sections from a water bath at 18 deg C, drying, soaking 1 min in benzene, drying, and then dipping again in NTB 3 emulsion. They were exposed at 4 deg C in a low-humidity, 100% CO/ sub 2/ atmosphere for 10 days, developed, and covered in the usual way. There was an average of 20.16 plus or minus 1.4 grains per labeled cell in the double emulsion group compared with 10.6 plus or minus 0.9 grains in the single emulsion group. In the double emulsion autoradiograph there were 55. 1 plus or minus 1.65 labeled cells per unit area as compared with 39.8 plus or minus 2.0 in the single emulsion group. In the double emulsion autoradiograph the grains were in 2 layers which could be seen simultaneously up to a magnification of 360. The relation between the photographic response of an emulsion to T-labeled cell nuclei in tissue sections is considered and discussed in terms of 2 main factors: the area of radioactive material in intimate contact with the emulsion, and the volume of radioactive material in useful relation to the emulsion. Since for BETA particles the maximum range in tissue is about 5.5 mu , only a few particles reach this distance and about 50% have been absorbed in the first 0.6 mu . Thus the larger the area of nuclear material which is in immediate contact with the emulsion, the greater will be the response. By using the double emulsion technique wifh sections of 1 mu and above, the response per nucleus follows closely the curve produced by comparing cut surface area per nucleus and section thickness, and this response will always be double that produced by a single emulsion layer for sections of the same thickness. (BBB)
- Research Organization:
- Royal Prince Alfred Hospital, Sydney
- Sponsoring Organization:
- USDOE
- NSA Number:
- NSA-18-011566
- OSTI ID:
- 4095943
- Journal Information:
- Stain Technology, Vol. 38, Issue 4; Other Information: Orig. Receipt Date: 31-DEC-64; ISSN 0038-9153
- Country of Publication:
- Country unknown/Code not available
- Language:
- English
Similar Records
Factors which Affect Efficiency of Autoradiography with Tritiated Thymidine
INTRACELLULAR LOCALIZATION AND QUANTITATION OF TRITIATED ANTIGENS IN RETICULOENDOTHELIAL TISSUES OF MICE DURING SECONDARY AND HYPERIMMUNE RESPONSES
Related Subjects
BENZENE
BETA PARTICLES
CARBON DIOXIDE
CYTOLOGY
EVAPORATION
HUMIDITY
LABELLED COMPOUNDS
MEASURED VALUES
METHACRYLATES
MICE
MICROSCOPY
NUCLEAR EMULSIONS
ORGANIC NITROGEN COMPOUNDS
RADIOACTIVITY
RADIOAUTOGRAPHY
RADIOSENSITIVITY
TEMPERATURE
THICKNESS
THYMIDINE
TRACER TECHNIQUES
TRITIUM
WATER